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P Lollar

Publications and source records attributed to P Lollar.

65 records · Page 4Linked to original sources
PubMed1984

Stabilization of thrombin-activated porcine factor VIII:C by factor IXa phospholipid.

The activation of porcine factor X by an enzymatic complex consisting of activated factor IX (factor IXa), thrombin-activated factor VIII:C (factor VIII:Ca), phospholipid vesicles, and calcium was studied in the presence of an irreversible inhibitor of factor Xa, 5-dimethylamino-naphthalene-1-sulfonyl-glutamyl-glycyl-arginyl- chloro met hyl ketone ( DEGR -CK). The formation of factor Xa was measured continuously by monitoring the increase in solution fluorescence intensity that occurs upon formation of DEGR -factor Xa. Omission of any component from the enzymatic complex reduced the reaction rate to a negligible level. In the presence of fixed excess factor IXa, the velocity of factor X activation was linearly dependent on the concentration of factor VIII:C, and thus, provided a plasma-free assay of factor VIII:C. Activation of factor VIII:C by 0.1 NIH U/ml thrombin in the presence of factor IXa, phospholipid vesicles, and calcium, followed at variable time intervals by the addition of factor X and DEGR -CK, was complete within 5 min, as judged by the fluorometric assay, and resulted in little or no loss of factor VIII:C activity over a period of 20 min; whereas, activation in the absence of either IXa or phospholipid vesicles decreased the half-life of factor VIII:C to approximately 5 min. Analysis of 125I-factor VIII:C-derived activation peptides by sodium dodecyl sulfate polyacrylamide gel radioelectrophoresis revealed identical results, regardless of whether factor IXa and/or phospholipid vesicles were included in the activation, suggesting that the lability of factor VIII:Ca is not due to a major alteration of its primary structure. We conclude that the activated porcine factor VIII:C molecule is stabilized markedly because of its interaction with factor IXa and phospholipid.

Amino Acid Chloromethyl Ketones↗
PubMed1981

Active-site-dependent, thrombin-induced release of adenine nucleotides from cultured human endothelial cells.

The effect of thrombin on release of adenine nucleotides is studied in cultured cell monolayers from the human umbilical vein. Thrombin-induced release of radioactivity from endothelial cells is dose-dependent and saturable with maximal response seen at 1 x 10(-8) M thrombin. The products are identified by thin-layer chromatography as adenine nucleotides. Diisopropylphosphoryl thrombin, which is enzymatically inactive, does not cause release of tritium. A 50-fold excess of diisopropylphosphoryl-thrombin, despite causing 98 percent inhibition of binding of 125I-thrombin to its high-affinity binding sites, does not inhibit thrombin-induced release. We conclude that (1) thrombin causes release from endothelial cells of adenine nucleotides and that (2) high-affinity, active-site-independent binding of thrombin is not involved in this process.

Adenine Nucleotides↗
PubMed1981

Massive fatal pulmonary emboli with fibrinolytic therapy.

A case is described in which massive fatal pulmonary emboli followed the initiation of thrombolytic therapy. Several important issues regarding anticoagulation and fibrinolytic therapy are addressed, including the possibility of anticoagulant failure and the risks of using fibrinolytic therapy in the setting of recent and remote thrombotic disease.

Adult↗
PubMed1981

Prostacyclin release from cultured and ex vivo bovine vascular endothelium. Studies with thrombin, arachidonic acid, and ionophore A23187.

Prostacyclin release from systemic and pulmonary endothelium has been evaluated in cultured cell monolayers and in an ex vivo vascular segment model in which the endothelium remains in contact with subendothelial structures. The effect of exposure to arachidonic acid, ionophore A23187, and thrombin on prostacyclin release has been assessed. Arachidonic acid elicited prostacyclin release in a dose-dependent fashion. Ionophore also stimulated bovine systemic endothelium to release prostacyclin. Thrombin-endothelial cell interactions have been examined extensively. Unlike umbilical venous endothelium, systemic and pulmonary bovine endothelium did not release prostacyclin following exposure to thrombin. Exposure to thrombin also failed to evoke the release of tritiated arachidonate metabolites from the bovine endothelial cell preparations. The presence of high affinity binding sites for thrombin (KD = 9.5 X 10(-9) M) on the bovine endothelium suggests that either thrombin binding is causally unrelated to prostacyclin release or that the bovine cells lack mediators required for thrombin to exert its effect.

Animals↗
PubMed1980

Binding of thrombin to cultured human endothelial cells. Nonequilibrium aspects.

The long-term incubation of 125I-thrombin with cultured human endothelial cells is studied and compared to the previously described rapid equilibrium binding in this system (Awbrey, B. J., Hoak, J. C., and Owen, W. G. (1979) J. Biol. Chem. 254, 4092-4095). Following rapid initial binding of thrombin, a much slower association of thrombin with endothelium is seen. As opposed to equilibrium binding, this process is 1) irreversible, 2) active-site specific, and 3) associated with covalent linkage to an Mr 30,000 protein. In addition this nonequilibrium binding appears unrelated to high-affinity equilibrium binding since blocking the high-affinity binding sites with diisopropylphosphoryl thrombin has no effect on nonequilibrium binding. The physiological role of this process is unknown.

Cells, Cultured↗
PubMed1980

Evidence that the effects of thrombin on arachidonate metabolism in cultured human endothelial cells are not mediated by a high affinity receptor.

The effect of thrombin and its derivative, diisopropylphosphoryl-thrombin on [3H]arachidonic acid metabolism is studied in cultured umbilical vein endothelial cell monolayers. Thrombin causes a dose-dependent release of radioactivity from endothelial cells fed [3H]arachidonate. Thin layer radiochromatography of acidified supernatants reveals that most of the radio-activity is [3H]arachidonate and its metabolites, 6-ketoprostaglandin F1 alpha and prostaglandin E2. Diisopropylphosphoryl-thrombin, which is enzymatically inactive, does not cause release of arachidonic acid or metabolites. A 50-fold excess of diisopropylphosphoryl-thrombin, despite causing 98% inhibition of binding of 125I-thrombin to its high affinity binding sites, does not inhibit thrombin-induced release. We conclude that the high affinity, active site-independent thrombin binding sites are not involved in thrombin-induced mobilization of esterified arachidonic acid.

Arachidonic Acids↗
PubMed1980

Clearance of thrombin from circulation in rabbits by high-affinity binding sites on endothelium. Possible role in the inactivation of thrombin by antithrombin III.

The clearance of (125)I-thrombin and diisopropylphosphoryl-(125)I-thrombin (DIP-thrombin) from the circulation in rabbits was studied. When given either intraarterially or intravenously, DIP-thrombin, which is active-site blocked, was approximately 90% cleared from the circulation by 1 min, the time of earliest sampling, indicating a large first-pass effect. DIP-thrombin given intravenously is found predominantly in the lungs, whereas DIP-thrombin injected into the aortic arch is distributed diffusely in approximate proportion to the blood supply. Renal artery, femoral artery, ear artery, left atrium, and portal vein infusions demonstrate that kidney, muscle, ear, heart, and liver, respectively, can remove DIP-thrombin from the circulation. These data imply that the clearance of DIP-thrombin is not a function of a specific organ but of the vascular bed per se. The clearance of DIP-thrombin was reversible since injection of 0.5 mg of unlabeled DIP-thrombin 10 min after the injection of a tracer dose of DIP-(125)I-thrombin resulted in the rapid reappearance of the DIP-(125)I-thrombin into the circulation. In addition, the clearance of DIP-thrombin was saturable, i.e., clearance of DIP-(125)I-thrombin was inhibited by unlabeled DIP-thrombin in a dose-dependent fashion. In vivo Scatchard analysis of the saturation of the clearance process demonstrated that DIP-thrombin can be removed by binding to high-affinity binding sites, since dissociation constants (K(D)) of 10 and 13 nM were obtained for human and bovine DIP-thrombin, respectively. In contrast to DIP-thrombin, approximately 75% of the radioactivity associated with active thrombin remained in the circulation at 1 min. By 10 min 55% of (125)I-thrombin had been removed from the circulation, and essentially all of the radioactivity can be accounted for in the liver. Sodium dodecyl sulfate-polyacrylamide gel radioelectrophoresis of plasma samples taken after injection of (125)I-thrombin demonstrated that all of the active thrombin was converted to covalent thrombin-antithrombin III complex by the time of initial sampling (30 s). The in vitro conversion of (125)I-thrombin to thrombin-antithrombin III complex was considerably slower (50+/-5% conversion at 30 s). The simultaneous injection of excess unlabeled DIP-thrombin inhibited the rate of formation of (125)I-thrombin-antithrombin III complex formation in vivo (but not in vitro), which suggests that the binding of active thrombin to the high affinity binding sites is required for the rapid inactivation of thrombin in vivo. We propose that (a) thrombin in the circulation binds to active site-independent high-affinity binding sites on the endothelial cell surface; (b) the inactivation of thrombin by antithrombin III is faster in vivo than in vitro because the high-affinity binding sites, present in a high concentration in the microcirculation, catalyze the reaction; (c) thrombin-antithrombin III complexes are selectively removed by the liver.

Animals↗