Biomedical subjects

P van de Waart

Publications and source records attributed to P van de Waart.

5 recordsLinked to original sources

PubMed1987

A functional test for protein S activity in plasma.

The physiological role of coagulation cofactor Protein S (PrS) for activated Protein C (APC) has recently been appreciated by the description of patients with PrS-deficiency, suffering from thromboembolism. The present study introduces a one-stage clotting assay for the assessment of PrS functional activity in plasma samples. The assay procedure is based on a factor Xa-initiated clotting test utilizing a mixture of AL(OH)3-adsorbed substrate plasma and patient's plasma supplemented with purified prothrombin (0.15 microM) and APC (0.05 microM), with phospholipids and CaCl2. Owing to the varying concentration of PrS in the sample plasma, clotting times were prolonged up to 25 seconds in the presence of APC, whereas no prolongation occurred in its absence. The test procedure proved to be specific for PrS, since preincubation with monospecific antibodies against PrS abolished the prolongation of clotting time, while reconstitution of adsorbed plasma with purified PrS restored its cofactor activity completely. The functional assay showed an inter-assay and intra-assay variation in the normal range of 11.7% and 10.1%, respectively (n = 20). PrS activity in a group of unselected patients (n = 34), revealing no abnormalities in global coagulation tests, amounted to 95.8 +/- 16.5% (mean +/- S.D.) with a range from 67% to 136% when analyzed in comparison to a plasma pool constituted from healthy volunteers. Patients (n = 32) undergoing oral anticoagulant therapy presented 21.1 +/- 10.8% residual PrS-activity accompanied by a concomitant decrease in PrS-antigen levels to 69.9 +/- 21.2%. The assay described is sensitive, it can be performed on routine basis and allows the detection of patients with PrS-deficiency.

Adult↗

PubMed1987

The effect of factor Va on lipid dynamics in mixed phospholipid vesicles as detected by steady-state and time-resolved fluorescence depolarization of diphenylhexatriene.

We have monitored the thermotropic behavior of mixed dimyristoylglycerophosphoserine (Myr2GroPSer)/dimyristoylglycerophosphocholine (Myr2GroPCho) and Myr2GroPSer/dipalmitoylglycerophosphocholine (Pam2GroPCho) vesicles in the presence of blood-clotting factor Va, using 1,6-diphenyl-1,3, 5-hexatriene as a lipid probe. The Ca2+-independent interaction of factor Va with these vesicles caused a small increase (1-2 degrees C) in the phase transition temperature, regardless of whether Myr2GroPChe was the lower or higher-melting component of the mixed vesicles. The major effect of factor Va was to increase the polarization of diphenylhexatriene when the mixed vesicles were in the liquid crystalline phase. The protein did not change the anisotropy in the bilayer gel state. The increase in the polarization value above the transition temperature closely correlated with the amount of phospholipid-bound factor Va, as verified by a direct binding technique. In addition, we found that the affinity of factor Va for Myr2GroPSer/Myr2GroPCho and Myr2GroPSer/Pam2GroPCho greatly increased at temperatures above the transition temperatures. Time-dependent fluorescence anisotropy measurements of diphenylhexatriene embedded in vesicles in the liquid crystalline state give fluorescence decay curves which can best be fitted by two exponential functions with two rotational correlation times and a constant term. Vesicles composed of Myr2GroPSer exhibit more ordering than Myr2GroPCho vesicles. However, the order parameter of mixed vesicles composed of 40% Myr2GroPSer and 60% Myr2GroPCho (mol/mol) approached that of Myr2GroPCho. Factor Va dramatically increased the longer rotational correlation time of diphenylhexatriene embedded in mixed vesicles in the liquid crystalline state from 3.7 ns to about 17 ns. The second rank-order parameter increased only slightly, but the calculated steady-state anisotropy increased by twofold. These results indicate that the acidic phospholipid-dependent binding of factor Va to mixed vesicles has an ordering effect on the acyl chains of the acidic phospholipids in the outer layer, but leaves the bulk of the phospholipids, mainly phosphatidylcholine, unaltered. None of the factor-Va-induced alterations in the anisotropy parameters point to the occurrence of lateral phase separation.

Diphenylhexatriene↗

PubMed1984

Interaction of prothrombin with factor Va-phospholipid complexes.

The effects of factor Va and the phospholipid-binding fragment of factor Va [factor Va light chain (LC), Mr 80000] on the binding of prothrombin, factor X, and factor Xa to phospholipid vesicles are reported. Equilibrium binding experiments were performed that utilized large-volume vesicles, which can be removed from the bulk solution by centrifugation. Factor Va decreased the dissociation constant of the prothrombin-phospholipid complex 50-fold, from 2.0 X 10(-7) M to 4.0 X 10(-9) M. For the factor X-phospholipid complex the decrease was 60-fold (1.8 X 10(-7) M to 3.0 X 10(-9) M) and for factor Xa, 160-fold (1.6 X 10(-7) M to 1.0 X 10(-9) M). The ratios of moles of protein bound to moles of total added factor Va at saturation of phospholipid-bound factor Va indicate an 1:1 stoichiometric complex of either factor Xa, factor X, or prothrombin and phospholipid-bound factor Va. In the presence of factor Va LC, the dissociation constants of factor Xa- and prothrombin-phospholipid complexes were increased, while the maximal protein-binding capacities of the vesicles were not affected by factor Va LC. The data suggest a competitive interaction between factor Xa and factor Va LC binding as well as between prothrombin and factor Va LC binding at the phospholipid surface. From this, it is concluded that the phospholipid-binding fragment of factor Va alone does not serve as the binding site for interactions of factor Xa and prothrombin with factor Va.

Animals↗

PubMed1983

Interaction of bovine blood clotting factor Va and its subunits with phospholipid vesicles.

Thrombin-activated factor Va and factor Va subunit binding to large-volume vesicles was investigated by a technique based on the separation by centrifugation of phospholipid-bound protein from the bulk solution. This technique allows the direct measurement of free-protein concentration. It is concluded that the phospholipid binding site on factor Va is located on a basic factor Va subunit with Mr 80 000 (factor Va-LC). The effects of phospholipid vesicle composition, calcium concentration, pH, and ionic strength on the equilibrium constants of factor Va- and factor Va-LC-phospholipid interaction were studied. Factor Va and factor Va-LC binding to phospholipid requires the presence of negatively charged phospholipids. It is further demonstrated that the following occur: (a) Calcium ions compete with factor Va and factor Va-LC for phospholipid-binding sites. (b) The dissociation constant of protein-phospholipid interaction increases with the ionic strength, whereas the maximum protein-binding capacity of the phospholipid vesicle was not affected by ionic strength. (c) The dissociation constant for factor Va-phospholipid interaction depends on pH when the vesicle consists of phosphatidic acid. It is concluded that factor Va-phospholipid interaction is primarily electrostatic in nature, where positively charged groups on the protein directly interact with the phosphate group of net negatively charged phospholipids. The results suggest that factor Va, like factor Xa and prothrombin, has the characteristics of an extrinsic membrane protein.

Animals↗

PubMed1982

Factor Va-factor Xa interaction. Effects of phospholipid vesicles of varying composition.

The interaction between factor Xa and factor Va was investigated both in solution and in the presence of phospholipid vesicles with varying contents of phosphatidylserine. The binding parameters were inferred from the kinetics of prothrombin activation. Factor Xa and factor Va form in solution an equimolar complex with a dissociation constant of 3.3 X 10(-9) M. Phospholipid vesicles promote the formation of the factor Xa-Va complex. The Kd of complex formation is dependent on both the phospholipid concentration and the composition of the phospholipid vesicle. For the interaction between factor Xa and factor Va in the presence of phospholipid vesicles containing 40 mol % dioleoylphosphatidylserine (DOPS) and 60 mol % dioleoylphosphatidylcholine (DOPC), the Kd increases linearly with increasing phospholipid concentration. In the presence of 10 microM phospholipid (DOPS/DOPC, 40/60 mol/mol) Kd = 3 X 10(-11) M. When the mole percentage of DOPS in the phospholipid vesicles is lowered from 20 to 5 mol %, there is a gradual increase of the Kd. In the presence of 10 microM phospholipid vesicles containing 5 mol % DOPS and 95 mol % DOPC Kd = 2.8 X 10(-10) M. The Kd measured in the presence of phospholipid vesicles containing 5 mol % DOPS and 95 mol % DOPC is independent of the phospholipid concentration. Two models are discussed that can quantitatively explain the effect of phospholipid vesicles on the complex formation between factor Xa and factor Va. Studies on the effect of the polypeptides with Mr 80 000 and Mr 94000 of which factor Va is composed on the Kd of the factor Xa-Va complex suggest that factor Xa binding to factor Va requires a Ca2+-mediated interaction between the two polypeptides.

Animals↗