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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.

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.

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.

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...

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.