thromboplastin and Rocky-Mountain-Spotted-Fever

The vascular endothelial cell (EC) is a primary target of infection with Rickettsia rickettsii, the etiologic agent of Rocky Mountain spotted fever. Changes in gene transcription elicited by intracellular infection, including EC expression of the coagulation pathway initiator known as tissue factor (TF), may contribute to the vascular pathology observed during disease. Nuclear run-on analysis of uninfected and infected, cultured human endothelial cells revealed that the rate of TF mRNA transcription is enhanced more than twofold at 3 h following infection, thus coinciding with increased steady-state levels of TF mRNA. TF mRNA remained relatively unstable during infection, with a half-life of 1.6 h. The eukaryotic protein synthesis inhibitor cycloheximide did not block R. rickettsii-induced increase in TF mRNA levels and actually resulted in its superinduction, thus revealing that de novo synthesis of host cell protein was not prerequisite to this transcriptional response. Involvement of the transcription factor NF-kappaB in R. rickettsii-induced TF expression was demonstrated by using two unrelated inhibitors of NF-kappaB activation. The antioxidant pyrrolidinedithiocarbamate and the proteasome inhibitor N-tosyl-L-phenylalanine chloromethyl ketone blocked expression of TF mRNA and activity during infection. This study demonstrates that R. rickettsii infection results in transcriptional activation of the TF gene and that this response involves activation of the transcription factor NF-kappaB.

Topics: Cells, Cultured; Cycloheximide; Endothelium, Vascular; Gene Expression Regulation; Humans; NF-kappa B; Proline; RNA, Messenger; Rocky Mountain Spotted Fever; Thiocarbamates; Thromboplastin; Tosylphenylalanyl Chloromethyl Ketone; Transcription, Genetic

Tissue factor (TF) mRNA expression was measured by in situ hybridization in the endothelium of the intact human umbilical vein after infection with Rickettsia rickettsii. At 4 hours, R rickettsii organisms were clearly visible within approximately 70% of endothelial cells by immunocytochemical staining. Quantitation of TF mRNA expression revealed that the level within endothelial cells of the infected vein was significantly greater (3.7-fold, P < .0001) than that detected in uninfected endothelial cells. Serial sections of the umbilical cord vein were processed for in situ hybridization, and immunocytochemical staining and showed TF expression in those endothelial cells that contained R rickettsii organisms. Immunocytochemical staining for TF antigen at 6 hours was negative, but TF was clearly demonstrated within macrophages and fibroblasts of both control and infected umbilical cords. These studies demonstrate that the vascular endothelial cell, ex vivo, can be directly induced to express TF mRNA. This observation has not heretofore been clearly demonstrated except for in cultured endothelial cells. Since R rickettsii infection induces thrombotic vascular occlusions in patients with Rocky Mountain Spotted Fever, the results imply a potential role for endothelial cell TF in the pathogenesis of thrombotic disease.

Topics: Actins; Endothelium, Vascular; Gene Expression Regulation; Humans; Rickettsia rickettsii; RNA, Messenger; Rocky Mountain Spotted Fever; Thromboplastin; Thrombosis; Umbilical Veins; von Willebrand Factor

Rickettsia rickettsii infection results in numerous responses by cultured endothelial cells, among them a rapid, transient increase in steady-state levels of tissue factor mRNA (L.A. Sporn, P.J. Haidaris, R.-J. Shi, Y. Nemerson, D.J. Silverman, and V.J. Marder, Blood 83:1527-1534, 1994). In this study, production of interleukin-1 (IL-1) was measured during infection and its potential role in autocrine cell stimulation was investigated. A fivefold increase in levels of IL-1 alpha antigen was measured in cell lysate samples by enzyme-linked immunosorbent assay at 18 h of infection. The majority of IL-1 alpha remained cell associated, as no significant increase was detected in culture medium. No IL-1 beta antigen was detected in cell lysates or culture medium from either control or infected cultures. A dramatic increase in the levels of IL-1 alpha mRNA occurred following infection, as measured by reverse transcriptase PCR, which revealed the appearance of the expected 421-kb product with RNA extracted from cells infected for 4 h and no detectable product from control cell samples. The presence of functional, cell-associated IL-1 alpha activity in infected cells was confirmed, following disruption, by the ability of the infected cells to induce tissue factor expression in target endothelial cells. Such induction was eliminated by pretreatment of the disrupted cell samples with neutralizing antibodies against IL-1 alpha but not against IL-1 beta. To investigate whether endogenously produced IL-1 participates in the stimulation of tissue factor expression, neutralizing antibodies against IL-1 or the IL-1 receptor antagonist were added to culture medium during infection. Both anti-IL-1 alpha and the IL-1 receptor antagonist resulted in approximately 40% inhibition of tissue factor expression, thus implicating IL-1 alpha in autocrine cell stimulation.

Topics: Antibodies, Blocking; Base Sequence; Cells, Cultured; DNA Primers; Endothelium, Vascular; Gene Expression; Humans; Interleukin 1 Receptor Antagonist Protein; Interleukin-1; Molecular Sequence Data; Neutralization Tests; Rickettsia rickettsii; RNA, Messenger; Rocky Mountain Spotted Fever; Sialoglycoproteins; Thromboplastin

An acutely ill 4-year-old girl with Rocky Mountain spotted fever (RMSF) was found to have a coagulation inhibitor. This child had no serious bleeding manifestations and minimal hemorrhagic skin manifestations despite severe RMSF, concurrent thrombocytopenia, as well as the coagulation inhibitor. Hemostatic abnormalities that occur with RMSF as well as other infectious illnesses associated with coagulation inhibitors are reviewed.

Topics: Autoantibodies; Blood Coagulation Disorders; Blood Coagulation Factors; Blood Coagulation Tests; Child, Preschool; Female; Humans; Lupus Erythematosus, Systemic; Partial Thromboplastin Time; Rocky Mountain Spotted Fever; Thromboplastin

We studied the coagulation and complement systems during Rocky Mountain spotted fever in Macaca mulatta experimentally infected with Rickettsia rickettsii. Ninety-one percent of monkeys infected intravenously with a high dose (10(6) plaque-forming units [pfu]) and 56% of monkeys infected with low doses (10(-1)-10(2) pfu) of R. rickettsii died after two to four days of illness. With the onset of fever and rickettsemia, animals developed hyperfibrinogenemia, mild thrombocytopenia, prolonged prothrombin and activated thromboplastin times, and increased serum fibrin/fibrinogen degradation products (FDP). Rickettsemia, thrombocytopenia, and FDP were greater in fatally ill monkeys than in survivors. Hemolytic titers of the second and third components of complement were not depressed except in a single surviving monkey that developed peripheral gangrenous ecchymoses at a time when both rickettsemia and agglutinating antibody were present. Thus, although activation and consumption of complement may occur during Rocky Mountain spotted fever, the hemostatic disturbances in fulminant infections seem to be a direct effect of the infectious vasculitis.

Topics: Animals; Blood Coagulation Factors; Complement System Proteins; Fibrin Fibrinogen Degradation Products; Fibrinogen; Haplorhini; Macaca mulatta; Prothrombin; Rickettsia rickettsii; Rocky Mountain Spotted Fever; Thromboplastin

Topics: Blood Cell Count; Blood Coagulation Disorders; Blood Coagulation Factors; Blood Coagulation Tests; Blood Platelets; Blood Pressure; Child; Child, Preschool; Factor V; Fibrinogen; Heparin; Humans; Hypotension; Infant; Infant, Newborn; Prothrombin Time; Rocky Mountain Spotted Fever; Sepsis; Shock, Septic; Thrombocytopenia; Thromboplastin