calcimycin and Hemorrhage

Topics: Blood Coagulation Disorders; Calcimycin; Calcium; Female; Hemorrhage; Humans; Lymphocytes; Middle Aged; Phosphatidylserines

Studies were performed on a patient with a longstanding bleeding disorder whose major defects were impaired platelet prothrombinase activity in the absence of added factor Va, and a platelet factor V value that was either decreased or at the lower limit of normal when assayed on multiple occasions. In contrast, plasma factor V values were consistently normal. Unlike Scott Syndrome, in which platelet prothrombinase activity is decreased in both the presence and absence of added factor V, her platelets appeared to utilize added factor Va normally in supporting the generation of prothrombinase activity. These findings suggest an intrinsic defect in platelet factor V as the basis of her platelet prothrombinase defect. This defect appears to be different than that described in the Quebec platelet disorder (factor V Quebec). Immunoblot analyses of washed platelet lysates demonstrated a pattern of variably sized factor V molecules that was entirely similar to that observed in normal platelets, and both the heavy and light chains of her factor V after thrombin cleavage were of the same size as that observed in normal platelets. In addition, her platelet multimerin was normal and immunoblot analysis excluded the type of generalized granular protein defect and pathological proteolysis that has been suggested to explain the factor V defect in the Quebec platelet disorder. The findings in this patient thus suggest a new type of platelet factor V defect as the basis for the impaired capacity of her activated platelets to support prothrombinase generation. The findings further support an important role for platelet factor V in hemostasis.

Topics: Blood Coagulation Factors; Blood Platelet Disorders; Calcimycin; Dose-Response Relationship, Drug; Factor Va; Female; Hemorrhage; Humans; Kinetics; Thrombin; Thromboplastin

A significant increase in intracellular Ca(2+) is required to trigger the remodeling of the cell plasma membrane. Scott syndrome is an extremely rare inherited disorder of the transmembrane migration of phosphatidylserine toward the exoplasmic leaflet in blood cells. We have recently reported a reduced capacitative Ca(2+) entry in Scott cells [Martínez et al. (1999) Biochemistry 38, 10092-10098]. We have investigated here the links between defective phosphatidylserine exposure and Ca(2+) signaling in Scott cells by focusing on the Ca(2+) entry following the emptying of intracellular stores. After depletion of caffeine- or thapsigargin-sensitive stores, Ca(2+) entry was lower in Scott compared to control lymphoblasts. However, the simultaneous depletion of both types of stores restored a normal Ca(2+) influx across the plasma membrane in Scott cells and phosphatidylserine externalization ability was improved concomitantly with capacitative Ca(2+) entry. These observations point to the essential role of capacitative Ca(2+) entry in the control of phosphatidylserine exposure of stimulated cells.

Topics: Aged; B-Lymphocytes; Blood Coagulation Disorders; Caffeine; Calcimycin; Calcium; Calcium Signaling; Cell Membrane; Cells, Cultured; Egtazic Acid; Female; Hemorrhage; Herpesvirus 4, Human; Humans; Phosphatidylserines; Reference Values; Syndrome; Thapsigargin

A series of potent and highly selective time-dependent monocyclic beta-lactam inhibitors of human polymorphonuclear leukocyte elastase (PMNE, EC 3.4.21.37) is described. The intrinsic potency of these compounds, as exemplified by L-680,833 (k(inactivation)/K(i) of 622,000 M-1.s-1), is reflected at the cellular level where it inhibits generation of the specific N-terminal cleavage product A alpha-(1-21) from the A alpha chain of fibrinogen by enzyme released from isolated polymorphonuclear leukocytes stimulated with fMet-Leu-Phe with an IC50 of 0.06 microM. The inhibitory activity of L-680,833 is also apparent in whole blood stimulated with A23187, where it inhibits formation of A alpha-(1-21) and PMNE-alpha 1-proteinase inhibitor complex formation with IC50 values of 9 microM. Pharmacokinetic studies indicate that after oral dosing L-680,833 is bioavailable in rats and rhesus monkeys. This oral bioavailability is reflected by the inhibition (i) of tissue damage elicited in hamster lungs by intratracheal instillation of human PMNE and (ii) enzyme released from human PMN stimulated after their transfer into the pleural cavity of mice. The properties of L-680,833 allow it to effectively supplement the activity of natural inhibitors of PMNE in vivo, suggesting that this type of low-molecular-weight synthetic inhibitor could have therapeutic value in diseases where PMNE damages tissue.

Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; beta-Lactams; Biological Availability; Calcimycin; Cricetinae; Fibrinogen; Hemorrhage; Humans; Lactams; Leukocyte Elastase; Lung Diseases; Macaca mulatta; Mice; Molecular Sequence Data; Molecular Structure; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Pancreatic Elastase; Peptide Fragments; Phenylacetates; Rats; Structure-Activity Relationship

Endothelium-derived relaxing factor, now identified as nitric oxide (NO), is a labile humoral agent formed by vascular endothelial cells from L-arginine. NO mediates the action of substances that induce endothelium-dependent relaxation and plays a role in regulating blood pressure. In this study we investigated whether NO is involved in the pathogenesis of the bleeding tendency associated with renal failure. Rats with extensive surgical ablation of renal mass develop renal insufficiency due to progressive glomerulosclerosis. Like uremic humans, rats with renal mass reduction and uremia have a bleeding tendency that manifests itself by a prolonged bleeding time. We found that N-monomethyl-L-arginine (L-NMMA), a specific inhibitor of NO formation from L-arginine, completely normalized bleeding time when given to uremic rats. L-NMMA injection also increased ex vivo platelet adhesion but did not affect ex vivo platelet aggregation induced by adenosine diphosphate, arachidonic acid, and calcium ionophore A23187. The shortening effect of L-NMMA on bleeding time was completely reversed by giving the animals the NO precursor L-arginine, but not D-arginine, which is not a precursor of NO. It thus appears that NO is a mediator of the bleeding tendency of uremia.

Topics: Animals; Arginine; Bleeding Time; Calcimycin; Endothelium, Vascular; Hemorrhage; Male; Nitric Oxide; omega-N-Methylarginine; Platelet Adhesiveness; Platelet Aggregation; Rats; Rats, Inbred Strains; Uremia