Biomedical subjects

George M Willems

Publications and source records attributed to George M Willems.

7 recordsLinked to original sources

PubMed2006

Protein S multimers are generated in vitro and affect protein S structure-function analyses.

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.

Complement C4b-Binding Protein↗

PubMed2005

Quantitative determination of the binding of beta2-glycoprotein I and prothrombin to phosphatidylserine-exposing blood platelets.

The plasma protein beta2GPI (beta2-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 beta2GPI has an appreciable interaction with PS (phosphatidylserine)-exposing cell membranes. However, quantitative data on the binding of beta2GPI to PS-exposing cells under physiologically relevant conditions are scarce and conflicting. Therefore we evaluated the binding of beta2GPI to PS-expressing blood platelets. Flow cytometry showed that binding of beta2GPI is negligible at physiological ionic strength, in contrast with significant binding occurring at low ionic strength. Binding parameters of beta2GPI 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 microM was found for beta2GPI, with 7.4x10(5) binding sites per platelet. Under physiologically relevant conditions (120 mM NaCl and 3 mM CaCl2), binding of beta2GPI was not detectable (extrapolated K(d)>80 microM). Prothrombin binding (at 3 mM CaCl2) was much less affected by ionic strength: K(d) values of 0.5 and 1.4 muM 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 beta2GPI suggest that only a small fraction (<5%) of the binding sites on PS-exposing blood cells are likely to be occupied by beta2GPI. These findings are discussed in relation to the alleged (patho-)physiological functions of beta2GPI.

Binding Sites↗

PubMed2005

Anti-prothrombin IgG from patients with anti-phospholipid antibodies inhibits the inactivation of factor Va by activated protein C.

Interference of anti-phospholipid antibodies with the protein C pathway has been suggested to play a role in the development of thrombosis in the anti-phospholipid syndrome. We studied the effect of IgG preparations containing anti-prothrombin antibodies of 17 lupus anticoagulant-positive patients and 12 controls on the inactivation of factor Va (FVa) by activated protein C (APC) in a system with purified coagulation factors. Test IgG was incubated with human prothrombin, phospholipid vesicles and CaCl(2). Protein S, FVa and APC were added and the APC-dependent loss of FVa activity was monitored over time. The residual amount of FVa remaining after 10 min was 14 +/- 4% (mean +/- SD) when 1.5 mg/ml normal IgG was present and ranged between 17% and 82% with 1.5 mg/ml patient IgG. Twelve patients IgG gave values of residual FVa >22% (i.e. 2 SD above the mean of controls), indicating that APC-mediated inactivation of FVa was significantly inhibited. The inhibition was strictly dependent on the presence of prothrombin, proportional to the concentration of IgG and strongly diminished at a 20-fold higher phospholipid concentration. Most, although not all, IgG containing anti-prothrombin antibodies inhibit the APC-catalysed FVa inactivation, which may contribute to the increased risk of thrombosis in patients with the anti-phospholipid syndrome.

Antibodies, Antiphospholipid↗

PubMed2004

The effect of phospholipids on the formation of immune complexes between autoantibodies and beta2-glycoprotein I or prothrombin.

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 beta2-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 beta2-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 beta2-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.

Animals↗

PubMed2003

Thrombogenicity of polysaccharide-coated surfaces.

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.

Adsorption↗

PubMed2002

Kinetics of prothrombin-mediated binding of lupus anticoagulant antibodies to phosphatidylserine-containing phospholipid membranes: an ellipsometric study.

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.

Antibodies, Antiphospholipid↗

PubMed2002

Fibrinogen adsorption, platelet adhesion and thrombin generation at heparinized surfaces exposed to flowing blood.

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.

Adsorption↗