George M. Rodgers

Preeclampsia is associated with a serum factor cytotoxic to human endothelial cells

Preeclampsia occurs in 7% to 10% of pregnancies and is a leading cause of morbidity for mothers and their infants. Intensive investigation has failed to reveal the cause of the multiple organ dysfunction characteristic of this disorder, which abates completely with delivery. However, several observations suggest that endothelial cell dysfunction is a central pathophysiologic event. We report that serum from preeclamptic women is cytotoxic to endothelial cells in vitro. Consistent with the reversal of the clinical condition after delivery, cytotoxic activity in serum of preeclamptic women is reduced after 24 to 48 hours post partum. In contrast, cytotoxic activity of serum from normal pregnant women increases after delivery.

Mitogenic activity is increased in the sera of preeclamptic women before delivery

There is increasing evidence that endothelial cell injury and altered endothelial cell function play an important role in the pathogenesis of preeclampsia. Endothelial cell injury can lead to the secretion of potent mitogens by activating platelets and directly through the increased production of peptide growth factors by endothelial cells themselves. This study was undertaken to test the hypotheses that increased secretion of mitogenic factors is a feature of preeclampsia and that this activity could be detected in the serum of preeclamptic women. Paired serum samples were collected in early labor and again at 24 to 48 hours post partum from term patients with preeclampsia (n = 15) and normal pregnant controls (n = 14). A bioassay was used to quantify mitogenic activity in these paired samples by assessing their ability to stimulate the incorporation of tritiated thymidine into deoxyribonucleic acid of confluent, quiescent (GO stage) human fibroblasts in monolayer culture. Mitogenic activity was significantly increased in prepartum, preeclamptic sera compared with normal controls and diminished rapidly postpartum to levels equivalent to normal pre- and postpartum serum. These findings are consistent with endothelial cell injury, a process that we believe plays a central role in the pathophysiology of the preeclamptic syndrome.

Characterization of the interaction between factor Xa and bovine aortic endothelial cells

Cultured bovine aortic endothelial cells incubated with Factor Xa activate prothrombin. Factor V, synthesized by the endothelial cells, or plasma Factor V and calcium are required for the reaction. In the present study, it has been demonstrated that 125I-Factor Xa binds specifically to endothelial cells. In addition, the activation of prothrombin by Factor Xa and aortic endothelial cells has been further characterized. The binding of 125I-Factor Xa to endothelial cells was saturable and reversible. The equilibrium dissociation constant (Kd) for 125I-Factor Xa binding was 3.6 X 10(-9) M, with 39000 molecules bound per cell. 125I-Factor Xa, inactivated by diisopropylfluorophosphate did not bind specifically to endothelial cells, indicating that the active site of Factor Xa was required for binding. Factor Xa, but not activated protein C, competed with 125I-Factor Xa for binding. Autoradiograms of sodium dodecyl sulfate-polyacrylamide gels of cell lysates indicated that the radiolabeled material that bound to the cells had electrophoretic mobility identical to Factors Xa alpha and Xa beta. Although Factor X partially inhibited the binding of 125I-Factor Xa, Factor Xa did not inhibit the binding of 125I-Factor X, indicating that the zymogen and enzyme bound to different receptors. The relationship of the 125I-Factor Xa binding which was measured in these studies to aortic endothelial cell prothrombin activation is unclear since an anti-Factor V IgG blocked prothrombin activation but not Factor Xa binding. Additionally, 125I-Factor Xa binds to nonvascular cells; these cells do not activate prothrombin in the presence of Factor Xa. Moreover, the calcium requirements for each reaction and the saturation curves of 125I-Factor Xa binding and prothrombin activation differ. Although these data do not exclude a relationship between Factor Xa binding and prothrombin activation, the binding of 125I-Factor Xa to aortic endothelium measured in these studies may be related to a separate cellular function. To further characterize prothrombin activation by Factor Xa and endothelial cells, the rates of thrombin generation by intact bovine aorta or endothelial cells derived from this tissue were compared and were found to be equivalent. These data indicate that vascular endothelium may serve as a physiologic surface for hemostasis.

Activation of coagulation Factor V by calcium-dependent proteinase

Factor V is a key coagulation cofactor, regulating the rate of Factor Xa-catalyzed prothrombin conversion. Activation of Factor V markedly accelerates coagulation. This study describes a new class of Factor V activators, sulfhydryl proteinases. Of the enzymes studied, calcium-dependent proteinase was the most effective activator. Activation of Factor V by this enzyme was associated with cleavage of 125I-labeled Factor V to peptides distinct from those generated by previously described activators. Calcium-dependent proteinase-activated Factor Va peptides with molecular weights of 114,000 and 93,000 bound both to Factor Xa and to cultured endothelial cells. Calcium-dependent proteinase was identified in vascular endothelial cells, a tissue that also synthesizes Factor V. These findings suggest a previously unknown mechanism for cellular regulation of coagulation.

Vascular smooth muscle cells synthesize, secrete and express coagulation factor V

Expression of cellular procoagulant activity may be one of the more important responses to vascular injury. Because factor V, a coagulation cofactor in the prothrombinase complex, catalyzes the conversion of prothrombin to thrombin, it may be a key to understanding this response. Therefore, we have investigated the synthesis, secretion and expression of factor V by vascular smooth muscle cells, which proliferate at sites of vascular injury. Cultured aortic vascular smooth muscle cells constitutively secreted Factor V activity, as determined by a functional assay. Labeled factor V was immunoprecipitated from conditioned medium of [35S]methionine-labeled cells, indicating that the secreted factor V was synthesized by vascular smooth muscle cells. Treatment of vascular smooth muscle cells with tunicamycin prevented secretion of factor V, suggesting that its secretion was dependent on the presence of N-linked carbohydrate. Factor V activity was also expressed on the vascular smooth muscle cell surface, as indicated by the ability of cultured cells to promote factor Xa-catalyzed prothrombin activation. These data suggest that the proliferation of smooth muscle cells in response to vascular injury may be one mechanism that links vascular disease with thrombosis.

Blastic transformation of a well-differentiated monocytic leukemia: Changes in cytochemical and cell surface markers☆

The clinical course of a patient with a well-differentiated monocytic leukemia which later underwent blastic transformation is described. Cytochemical, ultrastructural and cell surface analysis data were obtained at periods throughout her illness and correlated with the blastic transformation. Although surface markers characteristic of monocytic leukemia persisted, a deficiency of peroxidase in the granules of this patients monocytes was observed as well as loss of alpha-naphthyl butyrate esterase staining during transformation.

Preeclampsia: An endothelial cell disorder

seven babies had positive blood cultures; antenatal infection had been suspected, but not proven, in one case only. Four mothers developed postpartum infection which responded to antibiotic therapy. All mothers with infection, presenting either antenatally or postnatally, delivered within 48 h of membrane rupture and in all infected neonates the duration of membrane rupture was 72 h or less. The occurrence of sepsis was not related to the gestational age at onset of the membrane rupture. We conclude that patients with rupture of the membranes of 72 h duration or longer may be admitted for expectant management, even if presenting early in the second trimester, without an increased risk of infection.

Enhancement of prothrombin activation on platelets by endothelial cells and mechanism of activation of factor V

Factor Xa-catalyzed prothrombin activation occurs on cellular surfaces, including those of vascular endothelial cells. In a reconstituted model, endothelial cells and platelets acted synergistically to maximally activate prothrombin in the presence of Factor Xa. Synergism was observed at platelet concentrations less than 1 X 10(8)/ml. Thrombin formation was required for optimal prothrombin activation by endothelial cells and Factor Xa, with thrombin serving as an activator of Factor V. These data provide additional support for the hypothesis that vascular endothelium is a physiologic surface for hemostasis.

Radioisotopic measurement of plasma kallikrein

Abstract A rapid and sensitive assay for plasma kallikrein has been developed using the radiolabeled tripeptide substrate N-benzoyl-L-prolyl-L-phenylalanyl-L-arginyl-( 14 C)-anilide. This assay can measure kallikrein in plasma at concentrations ranging from 25–1000 ng/ml. The assay does not require inactivation of kallikrein inhibitors.