Biomedical subjects

M L Pusey

Publications and source records attributed to M L Pusey.

23 records · Page 2Linked to original sources

PubMed1985

Studies on the procoagulant activity of human amniotic fluid. I. Stability and coagulation factor requirements.

A fraction of human amniotic fluid possessing procoagulant activity was purified 35-70 fold by gel filtration on Sepharose 4B. The activity eluted in the void volume indicating a particle size in excess of 5 X 10(6) daltons. The amniotic fluid factor (AFF) activity is stabilized on storage in the presence of Ca++ ions which could not be replaced by Mg++. Addition of phospholipids resulted in accelerated loss of activity. Steps taken to remove factor VII did not affect the activity, but factor X and V are required.

Amniotic Fluid↗

PubMed1984

Membrane binding properties of blood coagulation Factor V and derived peptides.

The interactions of factor V and factor Va light chain with phospholipid vesicles were compared. The results showed that the factor Va light chain bound with the same parameters as factor V when the proteins were present at similar densities on the membrane. The protein-vesicle collisional efficiency was 30-50% for both factor V and factor Va light chain. The factor Va light chain bound at a higher density, and the additional binding interactions had lower affinity. The dissociation process showed negative cooperativity, possibly due to competition for acidic phospholipids in the membrane. The higher molar packing density produced more rapid protein-membrane dissociation rate constants. However, when factor V and Va light chains were present at similar molar densities on the vesicle, the dissociation rates, estimated by two methods, were similar. Analysis of dissociation rates also showed that factor Va interacted with factor Xa on the membrane surface while factor Va light chain did not. Factor Va generated by thrombin digestion of factor V did not result in a major loss of membrane-bound protein mass unless ethylenenediaminetetraacetic acid was present; in the latter case the mass changes indicated that all peptides were removed from the membrane except factor Va light chain. Equilibrium and dynamic measurements showed that ionic strength had a major effect on the dissociation rate but not on the association process. The salt effect indicated interaction between oppositely charged species with the product of the number of charges equal to at least -5.5. Factor Va light chain appeared to interact with phospholipids via a general charge interaction rather than via a specific charge stoichiometry.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1984

Electron microscopy and hydrodynamic properties of blood clotting factor V and activation fragments of factor V with phospholipid vesicles.

The electron microscopic and hydrodynamic properties of factor V and factor Va-vesicle complexes were determined. Images of negatively stained factor V bound to vesicles showed the protein as a relatively large globular domain (9.5 nm diameter) connected to the membrane through a narrow protein region 0.5-3 nm in length. This connecting region was not always visible and was measured as the distance between the globular region and the apparent vesicle edge. Factor V protein alone usually appeared as two connected globular regions of 10.2 and 6.5 nm diameter. The two-domain protein structure appeared consistent with both the image of factor V alone and bound to the membrane. Factor V had no biological activity in a phospholipid-free prothrombinase assay system used. The proteolytically activated form of factor V generated by digestion with thrombin (factor Va) was at least 30,000 times more active. The electron microscopic images of factor Va-vesicle complexes showed a smaller protein that was more closely associated with the vesicle surface than was factor V. The light chain (Mr about 80,000) component of factor Va also bound to the surface of the vesicles and appeared to be largely external to the membrane. Protein-induced hydrodynamic radius changes for the factor V-vesicle and factor Va-vesicle complexes were 12.8 and 6.3 nm, respectively. The images observed in the electron microscope were used to calculate protein-induced radius changes. Comparison of these values with the experimentally determined hydrodynamic radius changes showed approximate agreement for factor Va-membrane complexes. However, the images of factor V-vesicle complexes suggested smaller hydrodynamic radius changes than were actually observed.

Animals↗

PubMed1983

The physical significance of Km in the prothrombinase reaction.

Key kinetic parameters for the prothrombinase complex formed on membranes of phosphatidylserine (PS)/phosphatidylcholine (PC) (40/60) (Km = 0.12 microM, kcat = 11 s-1) or PS/PC (2/98) (Km = 0.40 microM, kcat = 11 s-1) differed only slightly. In contrast, the density of proteins on the membrane surface at the km differed greatly for the two membranes. The kinetics appeared unaffected by conditions where the number of phospholipid vesicles (2% PS) exceeded the number of protein molecules. These results establish that the Km for the prothrombinase reaction is determined by the concentration of prothrombin in solution rather than its density at the membrane surface. This system can be treated as a dissociable enzyme acting on a soluble substrate.

Factor V↗

PubMed1982

Kinetic and hydrodynamic analysis of blood clotting factor V-membrane binding.

The kinetics and hydrodynamic properties of factor V-membrane interaction were characterized. Factor V bound to membranes containing acidic phospholipids with a high collisional efficiency. For membranes of 20% phosphatidyl-serine-80% phosphatidylcholine, an association rate constant of (1.13 +/- 0.10) X 10(8) M-1 s-1 was obtained. These membranes contained about 20 factor V binding sites per vesicle of 3.6 X 10(6) daltons. This association rate represented about a 30% collisional efficiency. Dissociation of factor V was measured by a fluorescence energy transfer method with a dissociation rate constant of 0.0055 s-1 at 10 degrees C. The equilibrium dissociation constant for binding to these membranes at 10 degrees C and 0.14 M ionic strength was 5 X 10(-11) M. Ionic strength, pH, calcium, and charge density in the membrane had large effects on the rate of factor V-membrane dissociation, indicating a strongly ionic interaction between protein and membrane. In contrast, the association rate was nearly insensitive to ionic strength. The membrane-binding properties were relatively unchanged after thrombin digestion of factor V or after long-term protein storage which resulted in loss of procoagulant activity. Other proteins of the prothrombinase reaction greatly decreased the rate of factor Va-membrane dissociation. At protein saturation, factor V increased the hydrodynamic radius of phospholipid vesicles by 11.4 nm. In contrast, factor Va increased the hydrodynamic vesicle radius by only about 5 nm. The mass of membrane-bound protein was comparable for both proteins.

Animals↗