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The plasma protein β2GPI (β2-glycoprotein I) has been proposed to mediate phagocytosis of apoptotic cells and to play a role in the antiphospholipid syndrome. This suggestion is based mainly on the presumption that β2GPI has an appreciable interaction with PS (phosphatidylserine)-exposing cell membranes. However, quantitative data on the binding of β2GPI to PS-exposing cells under physiologically relevant conditions are scarce and conflicting. Therefore we evaluated the binding of β2GPI to PS-expressing blood platelets. Flow cytometry showed that binding of β2GPI is negligible at physiological ionic strength, in contrast with significant binding occurring at low ionic strength. Binding parameters of β2GPI and (for comparison) prothrombin were quantified by ellipsometric measurement of protein depletion from the supernatant following incubation with platelets. At low ionic strength (20 mM NaCl, no CaCl2), a dissociation constant ( K d) of 0.2 μM was found for β2GPI, with 7.4×105 binding sites per platelet. Under physiologically relevant conditions (120 mM NaCl and 3 mM CaCl2), binding of β2GPI was not detectable (extrapolated K d>80 μM). Prothrombin binding (at 3 mM CaCl2) was much less affected by ionic strength: K d values of 0.5 and 1.4 μM were observed at 20 and 120 mM NaCl respectively. The low affinity and the presence of many lipid-binding proteins in plasma that can compete with the binding of β2GPI suggest that only a small fraction (

Antiphospholipid antibodies interact with phospholipid membranes via lipid binding plasma proteins, mostly, prothrombin and beta(2)-glycoprotein I. Using ellipsometry, we characterized prothrombin-mediated binding of lupus anticoagulant (LA) positive IgG, isolated from patients with antiphospholipid syndrome, to phosphatidylserine (PS)-containing membranes. LA IgG did not bind to membranes in the absence of prothrombin, but addition of prothrombin resulted in high-affinity binding of prothrombin-LA IgG complexes; half-maximal binding was attained at IgG and prothrombin concentrations of 10 microg/mL and 4 nM, respectively. Adsorption to membranes containing 10-40 mol % PS revealed that membrane-bound rather than solution-phase prothrombin determines the adsorption kinetics. Depletion of prothrombin and LA IgG from the solution results in rapid desorption which is strongly inhibited by addition of prothrombin but not of LA IgG. Prothrombin-mediated adsorption of monovalent Fab1 fragments prepared from patient LA IgG was negligible, indicating that monovalent interaction between prothrombin and LA IgG is weak. The kinetics of adsorption and desorption indicate that divalent binding of LA IgG to prothrombin at the lipid membrane occurs.

SummaryThrombus formation at an artificial surface in contact with blood is a complex process that encompasses accretion of platelets from flowing blood and fibrin deposition. Platelet adhesion and fibrin formation are intimately intertwined reactions that are triggered by different sets of surface adsorbed plasma proteins. To dissect the contribution of protein adsorption and platelet adhesion to thrombin formation, a coherent study was performed with non-coated (NC) and heparin-coated (HC) surfaces. Thrombin production in whole blood, platelet adhesion and protein adsorption were studied using an amidolytic thrombin assay, a dynamic platelet adhesion assay and ellipsometry, respectively. Thrombin generation in flowing whole blood exposed to HC surfaces was greatly diminished when compared with NC surfaces. However, separate platelet adhesion and protein adsorption studies with anticoagulated whole blood revealed that platelets do not adhere because fibrinogen is not available in the protein layer that was deposited during the perfusion. These findings indicate that the in vitro thrombogenicity of a material cannot be predicted from platelet adhesion and protein adsorption data when these measurements are performed with anticoagulated blood or platelet rich plasma. Preincubation of NC and HC surfaces with fibrinogen or 2000-fold diluted plasma resulted in similar amounts of surface-bound fibrinogen and mediated massive platelet adhesion from flowing whole blood. These results indicate that a) platelet adhesion correlates with the availability of surface-bound fibrinogen and b) NC and HC surfaces are indistinguishable with respect to protein (fibrinogen) adsorption and platelet adhesion. It is apparent that the heparinized surface used in our studies exerts its anti-thrombogenic properties by neutralizing locally formed thrombin and not by reducing fibrinogen-dependent platelet adhesion.

Protein S, the cofactor of activated protein C (APC), also expresses anticoagulant activity independent of APC by directly inhibiting prothrombin activation via interactions with factor Xa, factor Va, and phospholipids. In different studies, however, large variations in APC-independent anticoagulant activities have been reported for protein S. The investigation presented here shows that within purified protein S preparations different forms of protein S are present, of which a hitherto unrecognized form (5% of total protein S) binds with high affinity to phospholipid bilayers (K(d)1 nM). The remaining protein S (95%) has a low affinity (K(d) = 250 nM) for phospholipids. Using their different affinities for phospholipids, separation of the forms of protein S was achieved. Native polyacrylamide gel electrophoresis demonstrated that the form of protein S that binds to phospholipids with low affinity migrated as a single band, whereas the high-affinity protein S exhibited several bands that migrated with reduced mobility. Size-exclusion chromatography revealed that the slower-migrating bands represented multimeric forms of protein S. Multimeric protein S (5% of total protein S) appeared to have a 100-fold higher APC-independent anticoagulant activity than the abundant form of protein S. Comparison of purified protein S preparations that exhibited a 4-fold difference in APC-independent anticoagulant activity showed that the ability to inhibit prothrombin activation correlated with the content of multimeric protein S. Multimeric protein S could not be identified in normal human plasma, and it is therefore unlikely that this form of protein S contributes to the APC-independent anticoagulant activity of protein S that is observed in plasma.

We investigated the mechanism by which anti-prothrombin antibodies cause lupus anticoagulant (LAC) activity. Addition of affinity-purified anti-prothrombin antibodies from LAC-positive plasma samples (alpha-FII-LAC+) to normal plasma induced LAC activity. Upon increasing the phospholipid concentration, LAC activity was neutralized. Addition of purified alpha-FII-LAC+ to normal plasma strongly inhibited factor Xa formation. No inhibition was measured when alpha-FII-LAC+ were added to prothrombin-deficient plasma or when purified anti-prothrombin antibodies from LAC-negative plasma samples (alpha-FII-LAC-) were added. When a combination of prothrombin and alpha-FII-LAC+ was added to the purified clotting complex, a strong inhibition of factor Xa and IIa formation was seen. The alpha-FII-LAC+ alone or a combination of prothrombin and alpha-FII-LAC- did not show inhibition. Ellipsometry studies showed that, in the presence of alpha-FII-LAC+, the affinity of prothrombin for a phospholipid surface increased dramatically, whereas a much lower increase was observed with alpha-FII-LAC-. Our results show that complexes of prothrombin and anti-prothrombin antibodies with LAC activity inhibit both prothrombinase and tenase. The antibodies increase the affinity of prothrombin for the phospholipid surface, thereby competing with clotting factors for the available catalytic phospholipid surface, a mechanism similar to that of anti-beta2-glycoprotein I antibodies.

J Biol Chem 2001 Mar 9;276(10):7164-8 Related Articles, Books, LinkOut Regulation of platelet factor Va-dependent thrombin generation by activated protein C at the surface of collagen-adherent platelets.Briede JJ, Tans G, Willems GM, Hemker HC, Lindhout T.Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, 6200 MD Maastricht, The Netherlands.Recent studies have indicated that factor Va bound to activated platelets is partially protected from inactivation by activated protein C (APC). To explore whether this sustained factor Va activity could maintain ongoing thrombin generation, the kinetics of platelet factor Va-dependent prothrombinase activity and its inhibition by APC were studied. In an attempt to mimic physiologically relevant conditions, platelets were adhered to collagen type I-coated discs. These discs were then spun in solutions containing prothrombin and factor Xa either in the absence or presence of APC. The experiments were performed in the absence of platelet-derived microparticles, with thrombin generation and inhibition confined to the surface of the adherent platelets. APC completely inactivated platelet-associated prothrombinase activity with an overall second order rate constant of 3.3 x 10(6) m(-)1 s(-)1, which was independent of the prothrombin concentration over a wide range around the apparent K(m) for prothrombin. Kinetic studies on prothrombinase assembled at a planar phospholipid membrane composed of 25 mol % phosphatidylserine and 75 mol % phosphatidylcholine revealed a similar second order rate constant of inhibition (2.5 x 10(6) m(-1) s(-1)). Collectively, these data demonstrate that ongoing platelet factor Va-dependent thrombin generation at the surface of collagen-adherent platelets is effectively inhibited by APC. No differences were observed between the kinetics of APC inactivation of plasma-derived factor Va or platelet factor Va as part of the prothrombinase associated with, respectively, a planar membrane of synthetic phospholipids or collagen-adherent platelets.

Annexin V, an intracellular protein with a calcium-dependent high affinity for anionic phospholipid membranes, acts as an inhibitor of lipid-dependent reactions of the blood coagulation. Antiphospholipid antibodies found in the plasma of patients with antiphospholipid syndrome generally do not interact with phospholipid membranes directly, but recognize (plasma) proteins associated with lipid membranes, mostly prothrombin or beta(2)-glycoprotein I (beta(2)GPI). Previously, it has been proposed that antiphospholipid antibodies may cause thrombosis by displacing annexin V from procoagulant cell surfaces. We used ellipsometry to study the binding of annexin V and of complexes of beta(2)GPI with patient-derived IgG antibodies to beta(2)GPI, commonly referred to as anticardiolipin antibodies (ACA), to phospholipid bilayers composed of phosphatidylcholine (PC) and 20% phosphatidylserine (PS). More specifically, we investigated the competition of these proteins for the binding sites at these bilayers. We show that ACA-beta(2)GPI complexes, adsorbed to PSPC bilayers, are displaced for more than 70% by annexin V and that annexin V binding is unaffected by the presence of ACA-beta(2)GPI complexes. Conversely, annexin V preadsorbed to these bilayers completely prevents adsorption of ACA-beta(2)GPI complexes, and none of the preadsorbed annexin V is displaced by ACA-beta(2)GPI complexes. Using ellipsometry, we also studied the effect of ACA-beta(2)GPI complexes on the interaction of annexin V with the membranes of ionophore-activated blood platelets as a more physiological relevant model of cell membranes. The experiments with blood platelets confirm the high-affinity binding of annexin V to these membranes and unequivocally show that annexin V binding is unaffected by the presence of ACA-beta(2)GPI. In conclusion, our data unambiguously show that ACA-beta(2)GPI complexes are unable to displace annexin V from procoagulant membranes to any significant extent, whereas annexin V does displace the majority of preadsorbed ACA-beta(2)GPI complexes from these membranes.

SummaryAnionic phospholipid membranes have a dual role in blood coagulation: they are essential for the initiation and propagation as well as for the limitation and termination of the blood coagulation process. Patients with the anti-phospholipid syndrome (APS) carrying antibodies against complexes of anionic phospholipids and plasma proteins, show in vitro inhibited phospholipid dependent coagulation reactions, whereas in vivo the presence of these antibodies is associated with an increased risk of thrombosis. In this study we focussed on the effects of these anti-phospholipid antibodies on the regulation of TF-mediated factor Xa (FXa) generation in plasma. We hypothesized that anti-phospholipid antibodies interfere with the phospholipiddependent inhibition by tissue factor pathway inhibitor (TFPI) of TFinduced coagulation. Indeed, total-IgG, anti-cardiolipin-IgG (aCL) and anti-β2GPI-IgG, isolated from patient plasmas, all stimulated TF-induced FXa generation in normal plasma. This enhanced FXa generation was not observed when the patient’s IgG was depleted of anti-β2GPI-IgG or when normal plasma was depleted of β2GPI or TFPI. Taken together, these data indicate that antibodies to β2GPI, circulating in patients with APS, suppress TFPI-dependent inhibition of TF-induced coagulation, which results in an increased FXa generation.

The physiological inhibitor of tissue factor (TF)zfactor VIIa (FVIIa), full-length tissue factor pathway inhibitor (TFPIFL) in complex with factor Xa (FXa), has a high affinity for anionic phospholipid membranes. The role of anionic phospholipids in the inhibition of TFzFVIIacatalyzed FX activation was investigated. FXa generation at a rotating disc coated with TF embedded in a membrane composed of pure phosphatidylcholine (TFzPC) or 25% phosphatidylserine and 75% phosphatidylcholine (TFzPSPC) was measured in the presence of preformed complexes of FXazTFPIFL or FXazTFPI1‐161 (TFPI lacking the third Kunitz domain and C terminus). At TFzPC, FXazTFPIFL and FXazTFPI1‐161 showed similar rate constants of inhibition (0.07 3 10 8 M 21 s 21 and 0.1 3 10 8 M 21 s 21 , respectively). With phosphatidylserine present, the rate constant of inhibition for FXazTFPIFL increased 3-fold compared with a 9-fold increase in the rate constant for FXazTFPI1‐161. Incubation of TFzPSPC with FXazTFPIFL in the absence of FVIIa followed by depletion of solution FXazTFPIFL showed that FXazTFPIFL remained bound at the membrane and pursued its inhibitory activity. This was not observed with FXazTFPI1‐161 or at TFzPC membranes. These data suggest that the membrane-bound pool of FXazTFPIFL may be of physiological importance in an on-site regulation of TFzFVIIa activity.

In a previous report we demonstrated that the blood compatibility of poly(ether urethane) (PEU) was improved by grafting phosphorylcholine (PC) groups on the surface. The improved blood compatibility was indicated by decreased platelet adsorption/activation and reduced thrombin formation at the polymer surface in experiments in which the surfaces were contacted with platelet-rich plasma in vitro. In the present study, we investigated the effect of grafted PC groups at a PEU surface on protein and phospholipid adsorption. Adsorption of human fibrinogen (Fg), human serum albumin (Alb), human high-molecular-weight kininogen (HMWK), and dioleoyl phosphatidylcholine (DOPC) vesicles was measured by ellipsometry. For this purpose, thin PEU films were cast on silicon wafers. The polymer film was photochemically modified with a PC-containing aryl azide. The presence of PC groups on the polymer surface was demonstrated by ESCA (Electron Spectroscopy for Chemical Analysis). The hydrophilicity of the polymer surface increased by the surface modification, as indicated by a decrease of the contact angle from 59 degrees before to 43 degrees after modification. Our data show that the presence of PC groups has little effect on the adsorption of proteins to a PEU surface. The highest adsorption was observed for Fg (0.49 microgram/cm2 on PC-modified PEU and 0.50 microgram/cm2 on PEU), followed by HMWK (0.28 microgram/cm2 on both PC-modified PEU and PEU), and Alb (0.16 microgram/cm2 on PC-modified PEU and 0.18 microgram/cm2 on PEU). Protein adsorption was further studied on a "biomembrane-like" DOPC bilayer formed on hydrophilic silicon. We found no protein adsorption on this DOPC bilayer. The adsorption of small unilamellar DOPC vesicles on the polymer surfaces amounted to about 0.06 microgram/cm2 (corresponding to circa 30% of monolayer coverage) and was similar for PC-modified PEU and PEU. Despite this partial surface coverage, preadsorbed DOPC on the polymer surface diminished the subsequent adsorption of proteins considerably. These results show that the mere presence of phosphorylcholine groups on a PEU surface is insufficient to suppress protein adsorption. The highly ordered structure of natural phospholipid bilayers seems to be required to suppress protein adsorption effectively.

The interaction of tissue factor pathway inhibitor (TFPI), factor Xa, and TFPI−factor Xa complexes with negatively charged phospholipid membranes composed of 25 mol % phosphatidylserine and 75 mol % phosphatidylcholine was studied by ellipsometry. The binding of TFPI alone was negligible; factor Xa bound with moderate affinity, with a dissociation constant Kd = 42 nM. Formation of the TFPI−factor Xa complex drastically enhanced the affinity for phospholipid membranes, Kd = 5 nM, compared to that of either protein alone. TFPI1-161, a TFPI variant lacking the third Kunitz domain and the positively charged C-terminus did not enhance binding affinity of the factor Xa. Analysis of the kinetics of adsorption and desorption confirmed the equilibrium binding data, although upon longer residence at the lipid membrane the desorption rate of TFPI−factor Xa complexes became slower, indicating an increase in affinity with longer residence of the TFPI−factor Xa complexes at the membrane. In contrast, binding of TFPI−fa...

SummaryWe have made use of a novel flow reactor to study the initiation and propagation of the ex vivo blood coagulation processes at artificial surfaces. The flow reactor consisted of a primary glass or polymer capillary that is connected to a secondary glass capillary, which inner wall was coated with a phospholipid bilayer of 25 mol% dioleoylphosphatidylserine/75 mol% dioleoylphosphatidylcholine (DOPS/DOPC). Citrated platelet free plasma and a CaCl2 solution were delivered by syringe pumps and mixed just before the entrance of the flow reactor. The outflowing plasma was assayed for factor XIa, factor IXa, factor Xa and thrombin activity. Perfusion of recalcified plasma through a bare glass capillary resulted in a transient generation of fluid phase factor XIa. In contrast, factor IXa production increased slowly to attain a stable steady-state level. We established that surface-bound factor XIa was responsible for a continuous production of factor IXa. Factor IXa-induced generation of factor Xa and thrombin was only observed when contact activated plasma was subsequently perfused through a DOPS/DOPC-coated capillary, showing that propagation of the factor IXa trigger requires a procoagulant, phosphatidylserine-containing, phospholipid membrane. The negatively charged inner surface of a heparin-coated polyurethane capillary, generated like the glass capillary significant amounts of factor XIa and factor IXa when perfused with recalcified plasma. No differences were found between unfractionated heparin and heparin devoid of anticoagulant activity. Thus, it is concluded that contact activation and factor IXa generation in flowing plasma is not inhibited by immobilised anticoagulant active heparin. Consequently, factor IXa-dependent thrombin generation at a downstream located phospholipid membrane was similar, regardless the specific anticoagulant activity of immobilised heparin.

SummaryTissue factor : factor VIIa induced activation of blood coagulation is inhibited by the complex between factor Xa and tissue factor pathway inhibitor (factor Xa : TFPI). We recently reported that phospholipid-bound factor Xa reduces the high binding affinity of factor Xa : TFPI for negatively charged phospholipids by a partial degradation of TFPI (17). The present study was undertaken to elucidate the factor Xa cleavage sites in TFPI and to delineate the consequences of this proteolysis with respect to the inhibitory activity of factor Xa : TFPI. We found that phospholipid-bound factor Xa cleaves in TFPI the peptide bonds between Lys86-Thr87 and Arg199-Ala200. Interestingly, Arg199 is the P1 residue of the third Kunitz-type protease inhibitor domain. The fast cleavage of the Arg199-Ala200 bond results in a 50-70% reduction of the anticoagulant activity of factor Xa : TFPI, as determined with a dilute tissue factor assay, but is not associated with a diminished inhibitory activity of factor Xa : TFPI towards TF : factor VIIa catalyzed activation of factor X. On the other hand, the slower cleavage of the Lys86-Thr87 peptide bond was associated with both a diminished anticoagulant and anti-TF : factor VIIa activity. Dissociation of factor Xa from the cleaved TFPI was not observed. These data provide evidence for a dual role of factor Xa since it is the essential cofactor in the TFPI-controlled regulation of TF-dependent coagulation as well as a catalyst of the inactivation of TFPI.

It is hypothesized that immobilized heparin exerts a dual role in blood coagulation. On the one hand, the heparinized surface is because of its dense negative charge, thought to initiate the intrinsic pathway of blood coagulation. On the other hand, heparin is known as a potent anticoagulant drug. However, it remains to be seen how much contact-phase activation of factor XI contributes to thrombin formation and how this process is counterbalanced by which of the anti-protease activities of immobilized heparin. In the present study we examined the generation of factors XIa, IXa, and Xa, and thrombin in recalcified normal and antithrombin-depleted plasma exposed to polyacrylamide-graft polyurethane (PU) sheets modified by multipoint attachment of two different heparin species. One of them, HAH, contained the specific antithrombin binding sequence and the other one, NAH, had a low affinity for antithrombin and had no anticoagulant activity. Our data demonstrate that in contrast to PU, PU-NAH and PU-HAH are strong mediators of factor XIa and factor IXa formation in normal and antithrombin-deficient plasma. Interestingly, compared to PU-HAH and PU-NAH, thrombin formation was only slightly diminished in antithrombin-deficient plasma exposed to PU. In contrast, thrombin formation was dramatically delayed and diminished in normal plasma exposed to PU-HAH. These findings indicate that very low amounts of factor XIa apparently suffice to induce significant amounts of thrombin. In this sense, heparinized surfaces are highly thrombogenic, but our data also indicate that this activity is effectively counterbalanced by the anti-thrombin activity of the immobilized anti-coagulant species of heparin.

beta 2-Glycoprotein I (beta 2GPI) is an essential cofactor for the binding to lipids of anticardiolipin antibodies (ACA), isolated from patients with anti-phospholipid syndrome. We used ellipsometry to study the binding of beta 2GPI and the beta 2GPI-mediated binding of ACA to planar membranes composed of phosphatidylcholine (PC) and 5-20 mol % phosphatidylserine (PS). No binding of beta 2GPI was observed to neutral (PC) membranes. Maximal binding of beta 2GPI was 3.2-3.6 pmol.cm-2. Affinity decreased strongly with decreasing PS content; increasing the NaCl and CaCl2 concentrations also led to a decrease in affinity. At physiologic conditions (10 mol % PS, 120 mM NaCl, and 3 mM CaCl2), a Kd of 14 microM was observed. Binding constants were insensitive to the chemical composition of the negatively charged phospholipid headgroup. ACA (1.25-10 micrograms.mL-1) caused a 30-40-fold enhancement of beta 2GPI binding to PS/PC membranes (20 mol % PS), resulting in the binding of about 2 pmol.cm-2 divalent ACA-(beta 2GPI)2 complexes at 100 nM beta 2GPI. In the absence of beta 2GPI, binding of ACA was negligible. Ad- and desorption kinetics of ACA-beta 2GPI complexes indicate that the initial monovalent association of ACA to membrane-bound beta 2GPI is rapidly followed by formation of divalent ACA-(beta 2GPI)2 complexes. Experiments with monovalent Fab1 fragments of ACA showed no appreciable effect on the beta 2GPI binding to lipid, substantiating the notion that divalent interactions are essential for the high-affinity binding of ACA-beta 2GPI. The anticoagulant effect of ACA is rationalized by the observation that binding of ACA-beta 2GPI complexes to the PSPC membrane severely restricts the adsorption of blood coagulation factor Xa.

The basic C-terminus of Tissue Factor Pathway Inhibitor (TFPI) appears to be essential for its anticoagulant activity when tested in a diluted thromboplastin prothrombin time assay. Although the data reported so far have increased our knowledge about the C-terminus as a major binding site for heparin, lipoproteins and phospholipids, it is still unclear how this region of TFPI plays a role in its anticoagulant mode of action. We earlier reported that in the presence of phospholipid the rate of association of factor Xa with full length TFPI (FL-TFPI) is about 10-fold faster than with C-terminus truncated TFPI. This in turn makes that, in vitro, full length TFPI is a more potent inhibitor of tissue factor – factor VIla catalyzed factor X activation than truncated TFPI. Binding studies, utilizing an ellipsometer, revealed that in contrast to the complex of C-terminus truncated TFPI (TFPI1-161) and factor Xa, the FL-TFPI.factor Xa complex has a high affinity for negatively charged phospholipids. However, when examined in a tubular flow reactor containing tissue factor embedded in a phospholipid bilayer composed of 25 mol% phosphatidyl-serine/75 mol% phosphatidylcholine, no differences in the potency of FL-TFPI and TFPI 1–161 to inhibit factor X activation were found. The two variants of TFPI did show an interesting difference though. We found that the quaternary complex of TF.factor VIla.FL-TFPI.factor Xa was much more stable than the complex containing TFPI1-161. This difference could not be attributed to their different phospholipid-binding properties since the same difference in stability was found on membranes that contained only DOPC.

The activation of prothrombin is catalyzed by prothrombinase, a complex of factor Xa and factor Va assembled on a negatively charged phospholipid membrane, We used a tubular flow reactor to identify the relative contributions of factor Va, prothrombin, and the negatively charged phosphatidylserine to the assembly of prothrombinase. Perfusion of phospholipid-coated capillaries with a mixture of factor Xa, factor Va, and prothrombin resulted in a steady-state rate of thrombin production that increased with (i) the phosphatidylserine content of the phospholipid bilayer, (ii) the factor Va concentration, and, most interestingly, (iii) the prothrombin concentration of the perfusion solution, Incorporation of 20 mol % phosphatidylethanolamine, a phospholipid with poor ability to promote prothrombinase activity, into a 5 mol % phosphatidylserine membrane also increased the steady-state rate of thrombin production, Direct measurements of the amount of prothrombinase in the flow reactor demonstrated that increased catalytic activities were the result of an increased steady-state amount of membrane associated prothrombinase. Thus, similar turnover numbers of prothrombin activation (3100 min(-1)) were calculated, irrespective of the phosphatidylserine content of the membrane, We established for membranes with low phosphatidylserine content (

Polyacrylamide-grafted polyetherurethane sheets were modified by end-point and multipoint attachment of heparin. The surface-bound heparin was firmly attached. No release of heparin activity could be detected when the surface was rinsed at a wall shear rate of 2000 s-1. Uptake of antithrombin and thrombin inactivation were investigated under well-defined flow conditions by the use of a spinning device with an attached disk-shaped heparinized surface. It is demonstrated that the rate of thrombin inactivation at the antithrombin-heparin surface equals the maximal rate of transport of thrombin toward the surface when the surface coverage of antithrombin exceeds 10 pmol/cm2. This result indicates that a higher intrinsic catalytic efficiency of a surface does not necessarily result in a higher antithrombin activity. We varied the heparin content of the surfaces between 0 and 35 micrograms/cm2 by increasing the number of functional groups to which heparin could be covalently attached. The uptake of antithrombin increased with the heparin content of the surface, but the stoichiometry decreased from 2 to 0.5 pmol antithrombin/micrograms heparin. Apparently, antithrombin could not bind to heparins buried in the poly(acrylamide) layer. The rate of thrombin inactivation at surfaces with low heparin content (2 micrograms/cm2) fells below the transport limit of thrombin and became proportional with the heparin content of the surface. Although the contribution of surface-bound heparin to the neutralization of fluid-phase thrombin was found to be negligible compared with the effect of fluid-phase antithrombin at physiologic relevant concentrations, these heparinized surfaces markedly delayed the onset of thrombin generation in platelet-rich plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

SummaryTissue factor-factor VIIa catalysed activation of factor X and factor IX is inhibited by the complex of tissue factor pathway inhibitor (TFPI) and factor Xa. At present, no information is available as to what extent the kinetics of complex formation between TFPI and factor Xa during factor X activation contribute to the overall rate of inactivation of the factor X converting complex. We have determined the kinetic parameters of the individual reactions, i. e. factor X activation, formation of the TFPI-factor Xa complex, and inactivation of tissue factor-factor VIIa by the TFPI-factor Xa complex. We modelled the overall reaction by assuming a two-step reaction: factor Xa generated by tissue factor-factor VIIa forms a reversible complex with TFPI and in the second step this complex forms a reversible quaternary complex with tissue factor- factor VIIa. The validity of the model was demonstrated by analysis of factor Xa generation curves in the presence of TFPI. Independently determined constants for factor X activation (kcat= 12 s-1, Km = 70 nM) and inhibition of tissue factor-factor VIIa by TFPI-factor Xa complex (rate constant of inhibition of 1.1 × 108 M-1s-1) were used. The association rate constant of the formation of the TFPI-factor Xa complex was estimated by fitting the model to the data. The rate constants of association of the complex between factor Xa and the variants full length TFPI, TFPI 1-247 and TFPI1-61 were very close to the values determined independently in a kinetic study on the inhibition of factor Xa in the presence of phospholipids, namely 3.4 × 106 M-1s-1, 0.4 × 106 M-1s-1 and 0.3 × 106 M-1s-1, respectively. These results indicate that the factor Xa-dependent inhibition of tissue factor-factor VIIa-catalysed factor X activation by TFPI can be adequately described by the two-step reaction sequence. We found that phospholipids (25 mol % phosphat-idylserine/75 mol % phospatidylcholine) increased the rate constant of association with factor Xa for full length TFPI, but not for the C-ter- minus truncated TFPI. Our results further indicate that optimal inhibition of tissue factor-factor VIIa activity is obtained with full length TFPI because of the higher rate of TFPI-factor Xa complex formation.

The inhibition equilibrium and kinetics of association and dissociation of the binding of three types of recombinant tissue factor pathway inhibitor (TFPI), namely full-length TFPI, C-terminal-truncated TFPI, and TFPI without the third Kunitz domain (TFPI1-161), to factor Xa have been measured. Formation and dissociation of the complexes were monitored by continuous measurement of the changes in the rate of hydrolysis of a peptidyl-p-nitroanilide substrate. Progress curves of product formation were fitted to a set of equations describing a one-step bimolecular inhibitory reaction in the presence of a competing substrate. For full-length TFPI the rate constants of association (kon) and dissociation (koff) were (5.1 +/- 0.7) x 10(6) M-1.s-1 and (2.6 +/- 0.9) x 10(-4)s-1 respectively. Thus, although the inhibition constant (50 pM) is far below the plasma concentration (2.5 nM) of TFPI, the half-time for transition to equilibrium in plasma is rather long (66s). The truncated forms of TFPI differ in that they have a 4-fold lower kon value but a similar dissociation rate constant. Therefore the inhibition constant, Ki, is 4-fold higher (0.2 nM) and the half-time to achieve equilibrium is prolonged to 250 s. The kon values of full-length and C-terminal-truncated TFPI, but not that of TFPI1-161, were found to decrease with increasing ionic strength.

The kinetics of prothrombin adsorption on planar bilayers, prepared from 40% dioleoyl phosphatidylserine and 60% dioleoyl phosphatidylcholine, are studied by off-null ellipsometry. A simple expression for the adsorption rate is derived directly from measured adsorption rates. The interpretation of this expression in terms of transport processes shows that up to a surface coverage Γ = 1.8 pmol cm −2 , i.e. 50% of the maximal surface coverage Γ max , the adsorption rate appears to be regulated by diffusion-limited transport of prothrombin from the bulk solution to the adsorbing surface. The adsorption rate is proportional to the concentration difference between the bulk and the solution immediately adjacent to the surface. The latter concentration appears to be instantaneously in equilibrium with the surface excess, conforming to the classical Langmuir isotherm. For low surface coverages, below 50% of maximal adsorption, the kinetics are thus described by a simple model containing the mass transfer coefficient, the dissociation constant K d and the maximal surface coverage Γ max , With known Γ max this model allows the estimation of K d from a single adsorption curve. However, for surface coverages exceeding 60% of maximal adsorption, drastic deviations from this model are found. The intrinsic adsorption rate is substantially reduced, presumably by surface exclusion.

In most flow systems, the rate of protein transfer from bulk solution to a macroscopic surface is site-dependent. In studies on surface- mediated protein conversion, this hampers the comparison of a proposed expression for the conversion process, such as the Michaelis-Menten equation, which actually measured overall conversion rates. However, the rotating disc is a classical example of a uniformly accessible surface and therefore was used for a quantitative analysis of prothrombin conversion by the phospholipid-bound factor Xa/factor Va complex (prothrombinase complex). A simple design of a rotating disc, adapted for ellipsometric measurement of protein adsorption, is presented. Agreement between experiment and theory was obtained for the influence of rotation velocity on the initial, transport-limited, adsorption rates of lysozyme, prothrombin, and fibrinogen. After coverage of the disc with a 20% phosphatidylserine/80% phosphatidylcholine bilayer and preadsorption of factor Va, addition of excess factor Xa and prothrombin resulted in effective conversion of prothrombin. For high (10 fmol.cm-2) surface coverage of prothrombinase, the rate of conversion equals the transport limited adsorption rate of prothrombin. For low (0.1 to 0.5 fmol.cm-2) surface concentrations of prothrombinase, the conversion rate dropped below the transport limit and the intrinsic kinetic parameters could be estimated at Km = 7.1 +/- 1.2 nM and kcat = 25 +/- 1.0 s-1 (20 degrees C). At these low surface activities of prothrombinase, the effect of the rotation rate (6 to 225 rad.s-1) on prothrombin conversion could be explained by the rotation-rate dependent prothrombin transport. This indicates that the fluid shear rate has no drastic influence on the intrinsic kinetics of prothrombin conversion.

No Evidence for Enhanced Thrombin Formation through Displacement of Annexin V by Antiphospholipid Antibodies

Prothrombinase assembly was studied on macroscopic planar bilayers consisting of 20% dioleoyl-phosphatidylserine (DOPS) and 80% dioleoyl-phosphatidylcholine (DOPC). The dissociation constant for the binding of factor Xa to the bilayer, measured by ellipsometry, was K(d) = 47 +/- 8 nM (mean +/- S.D.) and this value was lowered to K(d) = 2.2 +/- 0.3 pM by preadsorption of factor Va. This latter value was determined from direct measurement of steady-state thrombin production. A comparable value of K(d) = 1.0 +/- 0.1 pM was found by repeating these experiments in suspensions of phospholipid vesicles, and it was verified that prothrombinase assembly was not influenced by the addition of prothrombin.Using a minute amount (0.094 fmol cm-2) of preadsorbed factor Va, it was found that the rate of prothrombinase assembly exceeds the rate of collisions between Xa molecules from the buffer and the sparse Va molecules on the bilayer. Apparently, factor Xa adsorbs first to the membrane and then associates rapidly with factor Va by lateral diffusion. The data indicate almost instantaneous equilibrium of this complex formation on the surface with a lower limit for the bimolecular rate constant of k(on) = 2.8 x 10(13) (mol/cm2)-1 s-1.In suspensions of small phospholipid vesicles, prothrombinase assembly is collisionally limited and the value of k(on) should be proportional to vesicle diameter. This was verified with a method for estimation of k(on) values from thrombin generation curves. Values of 0.36 x 10(9) and 1.6 x 10(9) M-1 s-1 were found for vesicles of 20-30- and 60-80-nm diameter, respectively.

A simulation model for the production of thrombin in plasma is presented. Values of the reaction rate constants as determined in purified systems are used and the model is tested by comparison of simulations of factor Xa, factor Va and thrombin generation curves with experimental data obtained in thromboplastin-activated plasma. Simulations of the effect of hirudin indicate that factor V is predominantly activated by thrombin and not by factor Xa. The model predicts a threshold value for the factor Xa production which, if exceeded, results in explosive and complete activation of prothrombinase. The dependence of this threshold value on different negative feedback reactions, e.g. the inactivation of thrombin and factor Xa by antithrombin III (+ heparin), is investigated. The threshold value, for control plasma in the range of 1–10 pM total factor Xa production, can be raised two orders of magnitude by accelerated inactivation of factor Xa and prothrombinase but is hardly affected by a tenfold increase in the rate of thrombin inactivation or by increased production of activated protein C. This latter effect, however, results in a more gradual input-response relation between factor Xa input and the extent of prothrombinase activation.

Production of thrombin by phospholipid-bound prothrombinase complexes has been described as being regulated by the prothrombin concentration in the buffer (free-substrate model) as well as by the concentration of prothrombin adsorbed to the phospholipid surface (bound-substrate model). We studied simultaneous adsorption and conversion of prothrombin on planar bilayers consisting of 20% dioleoylphosphatidylserine and 80% dioleoylphosphatidylcholine. A transport limitation in the conversion of prothrombin was prevented by using a very low (0.3 fmol cm-2) amount of prothrombinase on the bilayer. The Michaelis and catalytic constants thus found were Km = 5.8 +/- 0.7 nM and kcat = 33 +/- 1 s-1 (mean +/- S.D.). The apparent bimolecular rate constant Kcat/Km = 5.7 x 10(9) M-1 s-1 exceeds the theoretically maximal value for the free-substrate model. In contrast, kcat/Km is within the range expected for a diffusion-controlled bound-substrate model. A similar mechanism for prothrombin conversion in suspensions of phospholipid vesicles would imply increasing kcat/Km values for increasing vesicle diameter. This prediction was tested and a 3-fold increase in kcat/Km values was indeed found for vesicles 60-80 nm in diameter compared to vesicles of 20-30 nm diameter. It is concluded that thrombin production is dependent on protein fluxes rather than on protein concentrations.

Generation and inhibition of activated factor IXa was studied in factor XIa-activated plasma containing 4 mmol/L free calcium ions and 20 mumol/L phospholipid (25 mol% phosphatidylserine/75 mol% phosphatidylcholine). Interference of other (activated) clotting factors with the factor IXa activity measurements could be avoided by using a highly specific and sensitive bioassay. Factor IXa generation curves were analyzed according to a model that assumed Michaelis-Menten kinetics of factor XIa-catalyzed factor IXa formation and pseudo first order kinetics of inhibition of factor XIa and factor IXa. In the absence of heparin, factor IXa activity in plasma reached final levels that were found to increase with increasing amounts of factor XIa used to activate the plasma. When the model was fitted to this set of factor IXa generation curves, the analysis yielded a rate constant of inhibition of factor XIa of 0.7 +/- 0.1 min-1 and a kcat/Km ratio of 0.29 +/- 0.01 (nmol/L)-1 min-1. No neutralization of factor IXa activity was observed (the estimated rate constant of inhibition of factor IXa was 0). Thus, in the absence of heparin, the final level of factor IXa in plasma is only dependent on the initial factor XIa concentration. While neutralization of in situ generated factor IXa in normal plasma was negligible, unfractionated heparin dramatically enhanced the rate of inactivation of factor IXa (apparent second order rate constant of inhibition of 5.2 min-1/per microgram heparin/mL). The synthetic pentasaccharide heparin, the smallest heparin chain capable of binding antithrombin III, stimulated the inhibition of in situ generated factor IXa, but sevenfold less than unfractionated heparin (k = 0.76 min-1 per microgram pentasaccharide/mL). We found that free calcium ions were absolutely required to observe an unfractionated heparin and pentasaccharide-stimulated neutralization of factor IXa activity. Factor XIa inhibition (psuedo first order rate constant of 0.7 min-1) was not affected by unfractionated heparin or pentasaccharide in the range of heparin concentrations studied.

Vascular anticoagulant alpha (VAC alpha, annexin V) is a member of the family of calcium and phospholipid binding proteins, the annexins. The binding properties of VAC alpha to phospholipid bilayers were studied by ellipsometry. Adsorption was calcium-dependent and completely reversible upon calcium depletion. Half-maximal adsorptions to phospholipid bilayers consisting of 100, 20, 5, and 1% dioleoyl-phosphatidylserine (DOPS) supplemented with dioleoyl-phosphatidylcholine (DOPC) were reached at Ca2+ concentrations of 0.04, 0.22, 1.5, and 8.6 mM. These surfaces all showed the same maximal adsorption of 0.22 +/- 0.01 micrograms of VAC alpha/cm2 (mean +/- S.D.). The adsorption to bilayers containing more than 10% DOPS was independent of VAC alpha concentrations in the range of 0.5-100 nM. Dissociation constants for VAC alpha binding to these surfaces were estimated to be below 2 x 10(-10) M. No adsorption was observed on pure DOPC bilayers at a Ca2+ concentration of 3 mM. The ability to mediate VAC alpha binding to 20% DOPS/80% DOPC bilayers was highly specific for Ca2+. The use of other divalent cations resulted in decreased binding in the order Cd2+ greater than Zn2+ greater than Mn2+ greater than Co2+ greater than Ba2+ greater than Mg2+. Zinc ions had a synergistic effect on Ca2(+)-dependent VAC alpha binding. The Ca2+ concentration needed for half-maximal binding to cardiolipin, dioleoyl-phosphatidylglycerol, DOPS, phosphatidylinositol, phosphatidic acid, dioleoyl-phosphatidylethanolamine, and sphingomyelin increased in that order. Adsorption was independent of the overall surface charge of the phospholipid membrane.

Plasma activities of creatine kinase (CK) and alpha-hydroxybutyrate dehydrogenase (HBD) were measured after permanent coronary artery occlusion in the dog. Cumulative release of enzymes in plasma was calculated from these data by using a previously validated two-compartment model for circulating enzymes. Regional myocardial ischemia was measured by injection of radiolabeled microspheres. After 48 hours, the dogs were killed, and a detailed map of left ventricular enzyme activity was obtained from 108 tissue samples. Cumulative release into plasma of CK and HBD was 96 +/- 20% and 112 +/- 26%, respectively, of the total activities depleted from the heart (mean +/- SD, n = 11). The scatter in these values is inherent to the calculations, and it is concluded that both enzymes are recovered completely in plasma and, thus, can be used as quantitative markers of injury. Discrepancies between this result and earlier reports on the recovery of CK are only partly apparent and can be explained partly by underestimation of the elimination rate of CK from plasma, irregardless of tissue edema and incomplete extraction of enzyme activity from tissue.

SummaryThe activation of prothrombin by prothrombinase was investigated in a continuous flow system at 25 °C. A glass capillary, containing a continuous phospholipid bilayer attached to the interior surface, was first perfused with factor Va. The factor Va bound to the phospholipid surface functioned as sites for the formation of prothrombinase, when subsequently a factor Xa and prothrombin containing solution was perfused. Under the conditions used, steady-state rates of prothrombin activation were attained after 4 to 15 min. The rates of prothrombinase formation increased with increasing factor Xa concentrations and flow rates, which is compatible with the assembly of prothrombinase being dependent on the flux of factor Xa to the phospholipid-bound factor Va. As long as factor Xa and prothrombin were present in the fluid phase the assembly of prothrombinase was apparently irreversible; during at least 20 min no loss of activity occurred. The steady-state rate of prothrombin activation was dependent on the surface concentration of prothrombinase, at 1.0 ΜM prothrombin and a shear rate of 82 s−1 the average rate was 870 mol thrombin/min per mol prothrombinase. In contrast to test tube experiments it was observed that in this flow system, the formation of Α-thrombin is favoured above the formation of meizothrombin (des fragment 1).

Purified human protein S preparations contain small amounts of multimeric protein S. Protein S multimers are absent in plasma, suggesting that multimerization results from purification. Protein S multimers effectively inhibit phospholipid-dependent reactions at low phospholipid concentrations, and may therefore interfere during functional analysis of protein S. We have demonstrated that anion-exchange chromatography, as well as high ionic strength or low pH elution conditions used in immunoaffinity purification of protein S, induce protein S multimer formation. When protein S multimers were removed from protein S preparations by size-exclusion chromatography, multimers spontaneously reappeared in the protein S monomer fraction. In model systems, high phospholipid concentrations (50 micromol/L) completely abrogate the inhibitory effect of protein S multimers on prothrombinase complex activity. In addition, C4BP does not bind to protein S multimers. Thus, at low phospholipid concentrations, addition of C4BP to purified protein S will not affect the inhibitory activity of protein S multimers. In conclusion, to avoid misinterpretations during protein S structure-function analysis due to multimers present in purified protein S preparations, we recommend studying the anticoagulant activities of protein S either in plasma, where protein S is in its unmodified natural form, or at high phospholipid concentrations in model systems with purified proteins.

In the last decennium, it became clear that antiphospholipid antibodies found in patients with antiphospholipid syndrome (APS) are in fact antibodies against lipid-bound plasma proteins. The most frequently occurring antigens are β2-glycoprotein I and prothrombin, although several other lipid-bound plasma proteins have been reported as antigen for antiphospholipid antibodies. Both proteins bind to anionic phospholipids, mainly phosphatidylserine, which becomes exposed at the surface of activated platelets, apoptotic cells, or cell-derived microparticles. The binding of β2-glycoprotein I and prothrombin to these cell surfaces or to artificial lipid vesicles with comparable amounts of anionic phospholipids is rather weak. Antiphospholipid antibodies from patients are predominantly of low affinity regarding their interaction with β2-glycoprotein I or prothrombin in solution. In the presence of a suitable phospholipid surface, however, this interaction is strongly enhanced. There is now strong evidence that formation of bivalent, trimolecular immune complexes at the lipid membrane essentially contributes to the binding of these intrinsically low affinity patient antibodies. Depending on the affinity, the epitope specificity, and the polyclonality of a particular IgG preparation, multimeric structures of lipid-bound immune complexes may form a lattice with multiple interactions on the lipid (cell) surface. It is hypothesized that the functional activity, that is, the ability of antibodies to interfere with lipid-dependent reactions, not only depends on their affinity for the antigen, but also on their ability to form multiple interconnected bivalent trimolecular complexes at the lipid (or cell) surface. It is further proposed that the rate of desorption of immune complexes may present a better indicator for the functional properties of the antibodies than the amount of adsorbed immune complexes.

Heparinization of artificial surfaces has been proven to reduce the intrinsic thrombogenicity of such surfaces. The mechanism by which immobilized heparin reduces thrombogenicity is not completely understood. In the present study heparin-, alginic acid- and chondroitin-6-sulphate-coated surfaces were examined for protein adsorption, platelet adhesion and thrombin generation. The protein-binding capacity from solutions of purified proteins was significantly higher for heparin-coated surfaces when compared with alginic acid- and chondroitin sulphate-coated surfaces. Yet, when the surfaces were exposed to flowing plasma, only the heparinized surface adsorbed significant amounts of antithrombin. None of the surfaces adsorbed fibrinogen under these conditions, and as a result no platelets adhered from flowing whole blood. Our results indicate that protein adsorption and platelet adhesion from anticoagulated blood cannot be used to assess the thrombogenicity of (coated) artificial surfaces. Indeed, the thrombin generation potentials of the different surfaces varied remarkable: while non-coated surface readily produced thrombin, alginic acid- and chondroitin sulphate-coated surfaces showed a marked reduction and virtually no thrombin was generated in flowing whole blood passing by heparinized surfaces.

Thrombus formation at an artificial surface in contact with blood is a complex process that encompasses accretion of platelets from flowing blood and fibrin deposition. Platelet adhesion and fibrin formation are intimately intertwined reactions that are triggered by different sets of surface adsorbed plasma proteins. To dissect the contribution of protein adsorption and platelet adhesion to thrombin formation, a coherent study was performed with non-coated (NC) and heparin-coated (HC) surfaces. Thrombin production in whole blood, platelet adhesion and protein adsorption were studied using an amidolytic thrombin assay, a dynamic platelet adhesion assay and ellipsometry, respectively. Thrombin generation in flowing whole blood exposed to HC surfaces was greatly diminished when compared with NC surfaces. However, separate platelet adhesion and protein adsorption studies with anticoagulated whole blood revealed that platelets do not adhere because fibrinogen is not available in the protein layer that was deposited during the perfusion. These findings indicate that the in vitro thrombogenicity of a material cannot be predicted from platelet adhesion and protein adsorption data when these measurements are performed with anti-coagulated blood or platelet rich plasma. Preincubation of NC and HC surfaces with fibrinogen or 2000-fold diluted plasma resulted in similar amounts of surface-bound fibrinogen and mediated massive platelet adhesion from flowing whole blood. These results indicate that a) platelet adhesion correlates with the availability of surface-bound fibrinogen and b) NC and HC surfaces are indistinguishable with respect to protein (fibrinogen) adsorption and platelet adhesion. It is apparent that the heparinized surface used in our studies exerts its anti-thrombogenic properties by neutralizing locally formed thrombin and not by reducing fibrinogen-dependent platelet adhesion.

J Physiol 2001 Sep 15;535(Pt 3):625-35 Related Articles, Books, LinkOut Ragged spiking of free calcium in ADP-stimulated human platelets: regulation of puff-like calcium signals in vitro and ex vivo.Heemskerk JW, Willems GM, Rook MB, Sage SO.Department of Biochemistry, Maastricht University, The Netherlands. jwm.heemskerk@bioch.unimaas.nl1. Human platelets respond to agonists of G protein (G(q))-coupled receptors by generating an irregular pattern of spiking changes in cytosolic Ca2+ ([Ca2+]i). We have investigated the ADP-induced Ca2+ responses of single, Fluo-3-loaded platelets in the presence or absence of autologous plasma or whole blood under flow conditions. 2. In plasma-free platelets, incubated in buffer medium, baseline separated [Ca2+]i peaks always consisted of a rapid rising phase (median time 0.8 s) which was abruptly followed by a slower, mono-exponential decay phase. The decay constant differed from platelet to platelet, ranging from 0.23 +/- 0.02 to 0.63 +/- 0.03 s(-1) (mean +/- S.E.M., n = 3-5), and was used to identify individual Ca2+ release events and to determine the Ca2+ fluxes of the events. 3. Confocal, high-frequency measurements of adherent, spread platelets (diameter 3-5 microm) indicated that different optical regions had simultaneous patterns of both low- and high-amplitude Ca2+ release events. 4. With or without plasma or flowing blood, the ADP-induced Ca2+ signals in platelets had the characteristics of irregular Ca2+ puffs as well as more regular Ca2+ oscillations. Individual [Ca2+]i peaks varied in amplitude and peak-to-peak interval, as observed for separated Ca2+ puffs within larger cells. On the other hand, the peaks appeared to group into periods of ragged, shorter-interval Ca2+ release events with little integration, which were alternated with longer-interval events. 5. We conclude that the spiking Ca2+ signal generated in these small cells has the characteristics of a 'poor' oscillator with an irregular frequency being reactivated from period to period. This platelet signal appears to be similar in an environment of non-physiological buffer medium and in flowing, whole blood.

There is increasing evidence that endogenously generated aldehydes formed as a result of lipid peroxidation are involved in the pathophysiological effects associated with oxidative stress in cells and tissues. Malondialdehyde (MDA), a major product of lipid peroxidation, can modify amines present on the cell surface and thereby introduce negative charges that can affect the interfacial ionic layer. We show that lipid peroxidation of RBC generates MDA adducts that, similar to phosphatidylserine (PS), bind annexin V in a Ca(2+)-dependent manner. Like PS, these adducts also promote the "PS-dependent" prothrombinase assays, albeit to lower levels. These results indicate that annexin V binding cannot be used as an exclusive indicator of cell surface PS and raise the possibility that some phenomenon attributed to PS may, in fact, also involve aldehyde-lipid adducts.

The inhibition of prothrombinase by tissue factor pathway inhibitor (TFPI) has been studied in the presence and absence of prothrombin, The rate constant of association of prothrombinase with full-length TFPI was 2.1 x 10(7) M(-1). s(-1) and 0.05 x 10(7) M(-1). s(-1) for the reaction with C-terminus truncated TFPI (TFPI1-161). The rate constant of dissociation was 0.65 x 10(-4) s(-1) in both cases. The rate constant of inhibition of prothrombinase by TFPI1-161 was similar to that of solution-phase factor Xa. In contrast, phospholipids and factor Va enhanced the association rate of the reaction between factor Xa and full-length TFPI by approx. 20-fold. Although TFPI, and in particular the full-length variant of the molecule, is a potent inhibitor of prothrombinase (overall inhibition constant of 3 pM), we also found that prothrombin competed very effectively with TFPI for the active site of factor Xa in the prothrombinase complex. A 50% reduction of the rate constant of inhibition was measured in the presence of 4 nM prothrombin, i.e. 0.2% of the plasma concentration of prothrombin. The physiological significance of TFPI as an inhibitor of prothrombinase activity is thus questionable.

The kinetics of inhibition of prothrombinase during prothrombin conversion by antithrombin and antithrombin-heparin complexes was studied in a tubular flow reactor. Prothrombinase was assembled at a macroscopic phospholipid membrane, composed of 25 mol % phosphatidylserine and 75 mol % phosphatidylcholine, deposited on the inner wall of a glass capillary, by perfusion with a factor Xa- factor Va mixture. Measurement of thrombin production allowed estimation of the amount of prothrombinase present at the capillary wall. Perfusion with a mixture of prothrombin and antithrombin or antithrombin-heparin complexes caused a progressive decline of the prothrombinase activity. The rate of inactivation steeply decreased with increasing prothrombin concentrations, indicating competitive inhibition. Analysis of competitive inhibition data requires estimation of the time-dependent substrate concentration, C,, near the prothrombin converting surface using earlier developed transport theory (Billy, D., et al. (1995) J. Biol. Chem. 270, 1029-10341. It appears that the inhibition rate is proportional to the fraction of enzyme, K,/(Km -I- C,), not occupied by substrate. The value of K, of prothrombinase estimated from the dependence of the inhibition rate on the prothrombin concentration (K, = 2-3 nM) is in excellent agreement with the value estimated from the substrate conversion rate (K, = 3 nM). Therefore inhibition of prothrombinase by antithrombin and antithrombin-heparin complexes is fully competitive with the substrate: prothrombin. Our results show that prothrombinase assembled on macroscopic lipid surfaces by virtue of its low K, value is protected for inhibition due to highly effective competition of prothrombin with antithrombin for the active site of factor Xa. The activation of prothrombin to thrombin is a central reaction of the blood coagulation that finally results in the formation of a hemostatic plug (Jackson & Nemerson, 1980; Mann et al., 1990). Effective activation of prothrombin requires prothrombinase, the complex of the serine protease factor Xa and cofactor Va assembled on a phosphati- dylserine-containing phospholipid membrane (Hemker et al., 1967; Nesheim et al., 1979; Rosing et al., 1980). The spatial propagation of the coagulation activation reactions is re- stricted by the requirement of phosphatidylserine-containing membranes, in vivo presumably provided by activated platelets and vascular lesions (Rosing et al., 1985; Mann et al., 1990; Zwaal et al., 1992, Comfurius et al., 1994). Furthermore, the coagulation is down-regulated in time by several inactivation reactions. The cofactors Va and VIIIa are eliminated by activated protein C (Esmon, 1989) that becomes available during thrombin generation. The extrinsic pathway is inhibited by tissue factor pathway inhibitor (Rapaport, 1989). The serine proteases of the blood coagu- lation are primarily inhibited by antithrombin (Egeberg, 1965; Abilgaard, 1969). This protein, with a plasma concentration of about 2 pM, forms equimolar complexes with serine proteases. The principal targets of antithrombin are factor Xa and thrombin. Heparin, widely in use as anticoagulant drug, up to 1000-fold enhances the rate of inactivation of

Factor VIII is a plasma protein which plays an essential role in the coagulation system. When assembled with the enzyme Factor IXa on a phospholipid membrane, it functions as a cofactor in the enzyme complex that cleaves the zymogen Factor X to Factor Xa. We studied the binding of both Factor VIII and the Factor VIII light chain to planar phospholipid bilayers consisting of 25% dioleoylphosphatidylserine and 75% dioleoylphosphatidylcholine (PSPC) by ellipsometry. Equilibrium-binding studies revealed that both Factor VIII and its light chain bind with high affinity to PSPC bilayers. The binding affinity of Factor VIII, with a dissociation constant Kd of 0.24 nM, was comparable with that of the Factor VIII light chain (Kd 0.49 nM). Maximal binding was 2.3 mmol of protein per mol of PSPC for Factor VIII and 7.1 mmol of protein per mol of PSPC for the Factor VIII light chain. Adsorption kinetics of both Factor VIII and its light chain conformed to the classical Langmuir adsorption model yielding dissociation constants calculated from the rates of adsorption that were similar to those obtained by equilibrium-binding studies. In contrast, measurements of rates of desorption revealed a deviation from those expected for a single class of binding sites. The desorption rate of Factor VIII increased with increasing residence time on the lipid membrane. This indicates transition of Factor VIII to a configuration with a lower binding affinity. As this time-dependent change in affinity could affect the validity of the measurement of binding parameters, in particular equilibrium-binding determinations carried out on a long timescale, binding affinity was also estimated from adsorption kinetics at half-maximal surface coverage, a relatively rapid procedure for the determination of the affinity. A Kd of 0.087 nM was obtained under these conditions. Measurement of equilibrium binding to small PSPC vesicles, a system in which equilibrium is rapidly attained, resulted in similar binding parameters (Kd = 0.13 nM and a maximal binding of 2.8 mmol of protein per mol of PSPC). These data confirm the results of equilibrium binding to planar bilayers. Taken together, our results indicate that Factor VIII, by means of its 80 kDa light chain, binds to PSPC bilayers with a dissociation constant below the concentration of Factor VIII in plasma and therefore may readily bind to exposed phospholipid membranes under physiological conditions.

Thrombin production by the phospholipid-bound complex of blood clotting factors Xa and Va (prothrombinase) was studied in a tubular flow reactor. The inner wall of a glass capillary was coated with a phospholipid bilayer of 25% phosphatidylserine and 75% phosphatidylcholine. Prothrombinase was assembled on this bilayer by perfusion with a mixture containing an excess of factor Va (2 nM) and a limiting amount of factor Xa (1-100 pM), either in the absence or presence of prothrombin. The rate of assembly of prothrombinase in the presence of prothrombin appeared to be limited by the transfer rate of factor Xa to the phospholipid surface. A good agreement was found between the predicted mass transfer coefficient for factor Xa and the observed pre-steady state rate of thrombin production. The eventually obtained steady state rates of thrombin production were proportional to the prothrombin concentration and independent of the surface density of prothrombinase. The observed rate of thrombin production was in excellent agreement with the predicted mass transfer rate for prothrombin. Transport-limited prothrombin conversion was observed for prothrombinase densities exceeding 1 fmol/cm2, which corresponds to 0.05% occupation of available binding sites. The kinetic parameters of the reaction were determined at low prothrombinase densities (0.02-0.04 fmol/cm2). Even in this situation the Michaelis-Menten equation had to be corrected for substrate depletion near the catalytic surface. We hereto employed an accurate approximation of the mass transfer coefficient. The kinetic parameter k cat was 60 s -1 and the intrinsic K m had a surprisingly low value of 3 nM. Both parameters were not influenced by the wall shear rate.

The conversion of prothrombin into thrombin is an imperative step in the sequence of reactions leading to the formation of a hemostatic plug. This reaction is catalyzed by the prothrombinase complex, composed of factors Xa and Va, which is assembled on a phospholipid surface through Ca-mediated interactions with the lipid polar headgroups. In this paper we describe experiments indicative for a major role of the stereochemical configuration of phosphatidylserine in the binding of the prothrombinase complex to a phospholipid surface. Using two stereoisomers of phosphatidylserine, i.e., L-alpha-glycerophosphoryl-L-serine (PLS) and L-alpha-glycerophosphoryl-D-serine (PDS), we demonstrate that membranes containing PLS are appreciably more favorable than membranes containing PDS in promoting assembly of the prothrombinase complex and catalysis of prothrombin conversion. Ellipsometric analysis of the binding of factor Va and factor Xa to a surface composed of phosphatidylcholine and 10 mol % of either PLS or PDS reveals that the apparent Kd for factor Va increases about 25-fold when substituting PDS for PLS. For factor Xa a 5-fold increase in Kd was observed on replacing PDS for PLS. When PLS is replaced by phosphatidyl-beta-lactate (PLac), a phospholipid resembling PS but lacking the amino group, a similar decrease in prothrombinase activity is found as observed with PDS, implicating the importance of both the amino group and the stereoconfiguration of the serine moiety for the assembly of the prothrombinase complex.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

A chimeric protein was produced with the N-terminal domain (amino acids 1-45) of annexin I and the core of annexin V (amino acids 19-320). This protein, annexin IN-VC, has a similar Ca2+ requirement for binding to phospholipid bilayers of 20% phosphatidylserine (PS)/80% phosphatidylcholine (PC) as annexin V. In contrast to annexin V, this protein has a strong potency to aggregate phospholipid vesicles as is shown by turbidimetric measurements and cryo-electron microscopy. Ellipsometry was employed to study quantitatively the phenomenon of phospholipid vesicle adhesion to annexin IN-VC bound to a planar phospholipid bilayer. The amount of phospholipid vesicles bound by annexin IN-VC on the planar bilayer is proportional to its surface coverage and can be inhibited by coadsorption of annexin V on the planar bilayer or by shielding the phospholipid surface of the vesicles with blood coagulation factor Va. Annexin IN-VC, like annexin V, does not bind to pure PC bilayers, but its adsorption on anionic phospholipid bilayers brings about the capacity to bind pure PC vesicles. This suggests that annexin IN-VC generates or exposes after binding to anionic phospholipids another phospholipid binding site, that differs from the annexin V phospholipid binding site. Collectively, the data suggest that two-dimensional cluster formation of annexin IN-VC on a bilayer with anionic phospholipids is involved in vesicle adherence.

In studies on the binding of proteins to small unilamellar phospholipid vesicles (SUV), the concentration of unbound protein usually remains unknown, because the vesicles cannot be separated from the bulk solution. In the present study, this limitation was overcome by addition of a supported planar phospholipid bilayer to the cuvette containing a vesicle suspension. Ellipsometric measurement of the protein adsorption velocities on this bilayer allowed determination of the concentrations of unbound protein. At high protein concentrations the adsorption is rapidly completed and the usual null-ellipsometry is too slow to obtain well-defined initial adsorption rates. Therefore, an off-null technique was developed, allowing measurement of the adsorbed protein mass at time intervals of 20 ms. Binding of prothrombin and coagulation factor Xa was measured in SUV suspensions prepared from a 20% dioleoylphosphatidylserine (DOPS) and 80% dioleoylphosphatidylcholine (DOPC) phospholipid mixture. For prothrombin, a dissociation constant K d = 140 ± 27 nM (mean ± S.E.) and maximal surface concentration Γ max = (8.9 ± 0.8) · 10 −3 mole of protein per mole of lipid, were obtained. For factor Xa, these values were K d = 49.6 ± 6.3 nM and Γ max = (23.0 ± 1.4) · 10 −3 mole of protein per mole of lipid. These binding parameters are similar to those obtained earlier for planar bilayers. Apparently, the binding of factor Xa and prothrombin is not dependent on surface curvature.

Cardiac ischemia causes interstitial leakage of cellular enzymes followed by release of these enzymes into plasma. Quantitative interpretation of these data requires a specific circulatory model, and the performance of such models was investigated. Plasma activities of cardiac enzymes were measured for increasingly abrupt forms of ischemic heart injury in the dog: 1) permanent ligation of the left anterior descending coronary artery (LAD); 2) reperfusion after 2 h of ligation of the LAD; and 3) calcium-free perfusion of the LAD during 10 min (calcium-paradox injury). Release into plasma of a rapidly (41%/h) and a slowly (2.2%/h) catabolized enzyme was calculated from the plasma activities, using a detailed circulatory model with compartments for heart, plasma, muscle, skin, and viscera. The time course of cellular enzyme leakage into interstitial space in the heart was calculated from release into plasma and a range of reported values for transendothelial permeability. Simplification to one- and two-compartment models introduced, respectively, 10 and 2% error in calculated cumulative release. Considering the other sources of error, this implies adequate performance of the two-compartment model. Protein washout from the heart is strongly influenced by expansion of interstitial protein space with dead myocyte volume and depends on the microheterogeneity of necrotic tissue areas. Accelerated release of enzymes into plasma after reperfusion reflects accelerated cellular leakage rather than enhanced washout.

In 1985 we isolated a new vascular anticoagulant protein VAC alpha, now called annexin V, with a high binding affinity (Kd less than 10(-10) M) for phospholipids. Its anticoagulant effect was attributed to displacement of coagulation factors from the phospholipid membrane. The present study demonstrates that the inhibition of prothrombinase activity by annexin V strongly depends on the curvature of the membrane surface and on the calcium concentration. Half-maximal inhibition of prothrombinase on and binding of annexin V to small vesicles, composed of 20% phosphatidylserine and 80% phosphatidylcholine, requires 2-3 mM calcium. With large vesicles and planar bilayers considerably less calcium is required for inhibition of prothrombinase and for lipid binding. Half-maximal binding of annexin V to large vesicles and to planar bilayers occurs at 0.7 and 0.2 mM calcium, respectively. This seemingly confirms the displacement model. The displacement of coagulation factors, however, proved to be incomplete, with residual surface concentrations of factors Xa, Va, and prothrombin sufficient for effective production of thrombin. Cryoelectron microscopy revealed that annexin V binding to large vesicles caused planar facets, indicating the formation of large sheets of clustered annexin V. Apparently, the formation of these two-dimensional arrays is promoted by calcium and hampered by high surface curvature. It is speculated that the complete inhibition (greater than 99%) of prothrombinase activity by annexin V is caused by the reduced lateral mobility of prothrombin and factor Xa in rigid sheets of annexin V covering the membrane.

A simple model of the initiation of thrombin formation in plasma as a response to factor Xa generation was constructed. In this model factor Xa is considered as an input with a constant concentration. Substrate depletion and inactivation by activated protein C are neglected. The resulting linear model allows a closed form solution by standard methods. With values of the reaction rate constants, as determined in purified systems, this model predicts a highly explosive and complete activation of factor V and prothrombin as a response to any given (steady state) factor Xa concentration even in situations where prothrombinase and(/or) thrombin are rapidly inactivated. However, the time delay to rapid thrombin production becomes longer at lower factor Xa concentrations. Analysis of this time delay as a function of the factor Xa concentration indicates that the gain of the feedback loop of factor V activation by thrombin is so high that the contribution of factor V activation by factor Xa is relatively unimportant for factor Xa concentrations in the nanomolar range. It appears that the time lag is mainly determined by the gain of this feedback loop: similar proportional reductions of each of these reaction rates causes a similar effect. The effects of moderately enhanced inhibition rates of thrombin and prothrombinase on the time delay depend strongly on factor Xa concentration. Only a minor prolongation of the delay is predicted for factor Xa concentrations in the nanomolar range, but for factor Xa concentrations in the 1–10 pM range, the enhanced decay will cause considerable delays. Simultaneous reduction of the turnover rate of prothrombinase results in much larger delays for the entire range of factor Xa concentrations.