Gel electrophoresis of polystyrene particles in glutaraldehyde crosslinked polyvinyl alcohol.

Polystyrene sulfate and carboxylate particles (19-189 nm radius) were subjected to electrophoresis in glutaraldehyde crosslinked polyvinyl alcohol of molecular weight 25.000 and 650.000 Da at various concentrations. The degree of crosslinking is severely limited by the mechanical properties of the gels that deteriorate beyond a glutaraldehyde concentration which decreases with increasing polyvinyl alcohol chain length. The effective fiber radius of the short-chain and long-chain polymer fiber was 45 +/- 25 and 131 +/- 47 nm, respectively. Thus, these media do not significantly exceed the apparent fiber thickness of agarose, are more difficult to prepare--but are well-defined synthetic products rather than natural ones, and have the advantage of carrying no net charge and can therefore be expected to exhibit no electroendosmosis.

Factor V binding to human platelets: the role of divalent cations and of platelet preparatory methods.

The binding of bovine factor V to human platelets has been studied to ascertain the influence of divalent cations as well as the manner of preparation of platelet suspensions. The binding of factor V to platelets was inhibited by EDTA and EGTA and required calcium (optimum concentration 5mM) but not magnesium. Factor V bound to a single class of low affinity sites in unstimulated gel filtered platelets with a Kd of 3.42 x 10(-8)M. Approximately, 2,340 factor V molecules were bound per platelet. Stimulation with the calcium ionophore A23187 or ADP and fibrinogen and inhibition with prostaglandins E1 (PGE1) or prostaglandins I2 (PGI2) failed to alter these constants. However, washing platelets by repeated centrifugation even in the presence of inhibitors of platelet activation decreased the number of binding sites to 1200 without change in the kd. These studies demonstrate a requirement for calcium in the physiological range for factor V binding to platelets, establish that washed platelets bind fewer factor V molecules than gel filtered platelets, and indicate that stimulation or inhibition of platelet function does not affect factor V binding.

Coagulation factor V exists uncomplexed in bovine plasma.

Bovine coagulation factor V has been examined immunochemically to ascertain whether the coagulant polypeptide (h) with Mr = 290,000-330,000 is complexed in plasma with a second immunochemically distinct polypeptide (l2) of Mr = 400,000. Antiserum containing antibodies to h and l2 detects the l2 polypeptide eluting earlier than the h chain on gel filtration of plasma with either added calcium of EDTA, consistent with the behavior of a higher molecular weight noninteracting species. An immobilized monospecific antibody to l2 removes only the l2 polypeptide from a purified factor V preparation containing both h an l2. Moreover, while a monospecific antibody to the h chain was able to precipitate purified radioactively labelled h chain alone or mixed with plasma, the l2 antibody was unable to precipitate radioactively labelled h chain even after attempted recombination of the h chain with l2 present in plasma. These studies indicate that the l2 polypeptide is not complexed to the h chain in a purified system or in plasma and reinforce the conclusion that factor V is a single polypeptide chain uncomplexed in plasma.

Immunochemical studies of factor V of bovine platelets.

Immunochemical techniques were employed to investigate the molecular properties of bovine platelet factor V. An antiserum prepared against purified factor V rapidly inactivated platelet factor V, indicating that platelet factor V is antigenically related to plasma factor V. A reaction of identity between factor V in plasma and purified factor V was documented by immunodiffusion against an antibody to platelet factor V. When platelet factor V was extracted with Triton X-100 in the presence of protease inhibitors and subjected to immunoelectrophoresis against the antiserum to plasma factor V, a single antigenic component migrating toward the anode was observed. In the absence of protease inhibitors, following release by collagen or solubilization, platelet factor V appeared close to the origin suggesting proteolytic alteration. Platelet factor V released by collagen or extracted with Triton X-100 was activated by thrombin 7.4-fold and 4.5-fold respectively, compared to a 17-fold activation of factor V in plasma under identical conditions. The ability of thrombin to activate platelet factor V as well as the close correspondence of factor V activity and antigen released by collagen indicates that the molecule is largely in the unactivated form after release. A single component of molecular weight 270 000 was seen when platelet factor V released by collagen was immunoprecipitated and subjected to dodecylsulphate gel electrophoresis. Factor V coagulant assays and immunoelectrophoresis of subcellular fractions showed that platelet factor V is localized primarily in the alpha granules. Platelet factor V appears to be similar to, if not identical with, plasma factor V by the criteria of immunologic identity, similar electrophoretic mobility and virtually identical molecular weights.

Effect of fibrin degradation products and thrombin on fibrinogen synthesis.

The effect of intravenous administration of homologous fibrin degradation products and thrombin on fibrinogen synthesis was assessed in rabbits. The relative fibrinogen synthesis rate was calculated as a ratio of the amount of radiolabelled lysine incorporated into fibrinogen to the amount incorporated into albumin during the same measurement period. An increase in this ratio above control would indicate a relatively specific stimulation of fibrinogen synthesis as compared with albumin, which is not an acute-phase reactant. Injection of 45 mg of 'early' or 'late' fibrin degradation products failed to produce a significant increase in the relative fibrinogen synthesis rate, suggesting that fibrin degradation products play no feedback role in controlling fibrinogen synthesis. Infusion of small amounts of homologous thrombin (15--25 NIH u) was followed by a small but statistically significant elevation of the relative fibrinogen synthesis rate. This was not accompanied by any increase in the levels of fibrinogen degradation products in plasma, or by any decrease in plasma fibrinogen concentration, possibly suggesting that thrombin can stimulate fibrinogen synthesis by a mechanism independent of significant fibrinogenolysis or intravascular coagulation.