Fibrinolytic and coagulant responses to regional limb perfusions of tumor necrosis factor, interferon-gamma, and/or melphalan.

Regional limb perfusion with antineoplastic agents stresses the local vasculature in a variety of ways. However, by monitoring the perfusates from limbs treated with melphalan alone or with melphalan plus tumor necrosis factor (TNF) and interferon-gamma (IFN-gamma), we were able to distinguish the effect of the cytokines on the observed coagulant and fibrinolytic responses. We collected samples of effluent from a series of lower extremities that were perfused with the cytokines and/or melphalan as treatment for localized melanoma. Both regimens produced statistically significant evidence of coagulant and fibrinolytic activation. However, limbs receiving cytokines in addition to the melphalan responded with a sharper rise in tissue plasminogen activator (tPA) and plasmin (plasmin-antiplasmin complexes [PAP]) than limbs treated with melphalan alone. Evidence of thrombin formation (prothrombin fragment 1 + 2 [F1 + 2], thrombin-antithrombin complexes [TAT]) was also greater when the cytokines were included, although the response was delayed and less consistent than the fibrinolytic activation.

Heparin enhances endothelial cell von Willebrand factor content by growth factor dependent mechanisms.

Von Willebrand factor (vWf), a multimeric adhesive glycoprotein synthesized, stored, and secreted in megakaryocytes and endothelial cells, is normally found in plasma, platelets and subendothelium. While many substances mediate the release of vWf from endothelial cells, factors that enhance vWf synthesis and partitioning to its regulated pathway are currently unknown. We studied the effect of pharmacologic doses of heparin on the vWf content of endothelial cells. After a lag of 8 h and in the presence of crude or purified growth factor, heparin at doses between 0.25 and 2 U (1.4-11 micrograms)/ml, increased the content of high molecular weight vWf. The increased amounts of vWf were localized to Weibel-Palade bodies and extracellular matrix. Lower molecular weight highly sulfated heparin or heparin-like compounds were most active in growth factor dependent endothelial cell vWf expression. There was no clear importance of polysaccharide sequence or protein core.

Gamma-interferon modulates von Willebrand factor release by cultured human endothelial cells.

Endothelial cells (EC) synthesize and secrete von Willebrand factor (vWF), a multimeric glycoprotein required for normal hemostasis. Within human endothelial cells, vWF multimers of extremely high molecular weight are stored in rod-shaped organelles known as Weibel-Palade bodies. Inflammatory mediators, such as interleukin-1, induce in vitro a variety of procoagulant responses by EC, including the secretion of stored vWF. We postulated that other inflammatory mediators might act to balance this procoagulant reaction, thereby assisting in the maintenance of blood fluidity during immune activation. Both gamma-interferon (gamma-IFN) and tumor necrosis factor (TNF) were found to act independently and cooperatively to depress the stimulated release of vWF from EC. Analysis of stored vWF in either gamma-IFN and/or TNF-treated EC demonstrated a loss of high molecular weight multimers while immunofluorescent studies documented a loss of visible Weibel-Palade bodies. This suggests that gamma-IFN and TNF interfere with normal vWF storage. gamma-IFN acted in a dose-, time-, and RNA-dependent fashion, and its inhibition of vWF release was reversible with time. No effect of gamma-IFN on EC was noted when anti-serum to gamma-IFN was added. Unlike gamma-IFN, alpha-interferon did not effect EC vWF. Therefore, gamma-IFN and TNF may be important in decreasing vWF release during inflammatory or immunologic episodes.

Subendothelial matrix of cultured endothelial cells contains fully processed high molecular weight von Willebrand factor.

Von Willebrand factor (vWf) is an adhesive glycoprotein composed of identical subunits linked by disulfide bonds to form multimers of varying sizes. VWf is found in platelets and plasma, where it functions in the adhesion of platelets to exposed subendothelium. Subendothelial matrix contains vWf, but the multimeric composition of matrix vWf, its binding site in matrix, and the mechanism by which it is delivered to matrix are unknown. Using human umbilical vein endothelial cell (HUVE) cultures, we have partially characterized subendothelial matrix vWf. Matrix from HUVE was solubilized in sodium dodecyl sulfate and electrophoresed on 1.25% agarose gels. The extracted vWf was composed of extremely high molecular weight (HMW) multimers of vWf not normally found in plasma. The vWf content of HUVE supernatant, extract, and matrix was quantitated and characterized by radioimmunoassay, agarose gel electrophoresis, and densitometry. The matrix-bound or -associated vWf represented 4% to 18% of total HUVE vWf. Western blot analysis of matrix vWf after reduction showed a subunit species of 220 kd. Long-term incubation of HUVE with phorbol 12-myristate 13-acetate (PMA), a compound that causes release of HMW vWf from Weibel-Palade bodies in HUVE, resulted in a marked decrease in matrix vWf (0.2% to 1% of the total vWf). The multimeric pattern of the remaining matrix vWf continued to show predominantly HMW multimers.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibody and immune complexes induce tissue factor production by human endothelial cells.

Patients with systemic lupus erythematosus (SLE) have an increased incidence of arterial and venous thromboses. The mechanism by which thromboses develop in these patients is unknown. We had previously observed that the sera of patients with SLE contain antibodies and immune complexes that can bind to endothelial cells. Because endothelial cells can synthesize tissue factor, a potent activator of coagulation, we studied the effect of IgG complexes and sera from patients with SLE on the production of tissue factor by these cells. Human umbilical venous endothelial cells incubated with heat-aggregated IgG (HA-IgG) (0.5 to 4.0 mg) elaborate procoagulant activity in a dose-dependent manner. All procoagulant activity was found in the particulate cell fraction, and none was secreted into the medium. Maximum expression of procoagulant activity required 6 to 8 hr, and its production was totally inhibited by the addition of cyclohexamide or actinomycin D. The presence of gel-filtered platelets augmented production of procoagulant activity by endothelial cells stimulated by HA-IgG. Endothelial cell procoagulant activity was not inactivated by diisofluoropropylphosphate, required the presence of Factor VII for its expression, and was neutralized by a specific anti-tissue factor antibody. Endothelial cells incubated with sera from 14 of 16 patients with SLE produced increased amounts of tissue factor compared with 21 normal sera (p less than 0.025). Fractions of two SLE sera containing monomeric IgG, IgA, or IgM, as well as fractions containing IgG complexes, each stimulated endothelial cells to produce more tissue factor than similar fractions prepared from two normal sera. These studies demonstrate that endothelial cells will produce the procoagulant tissue factor after exposure to anti-endothelial cell antibodies or IgG-containing immune complexes. The production of tissue factor by endothelial cells at sites of immune vascular injury may play a role in the development of thromboses in patients with SLE.

Differential effects of phorbol esters on normal myeloid precursors and leukemic cells: basis for autologous bone marrow reconstitution in acute nonlymphocytic leukemia using phorbol ester-treated bone marrow from patients in remission.

The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induces macrophage-like differentiation of HL60 cells and cells from patients with acute nonlymphocytic leukemia (ANLL). We assessed the use of TPA as a means of eradicating residual leukemia from remission bone marrow prior to autologous bone marrow reconstitution. A 30-min incubation with TPA led to marked growth arrest in HL60 cells and in cells from most patients with acute myelogenous leukemia and acute myelomonocytic leukemia, whereas cells from most patients with acute promyelocytic leukemia and acute undifferentiated leukemia demonstrated a lesser degree of growth arrest. Freezing and thawing, a necessary step in autologous reconstitution, had no effect on the cessation of proliferation induced in HL60 or ANLL cells preincubated with TPA for 30 min. Virtually normal myeloid precursor growth occurred in normal or remission bone marrow cells preincubated with TPA and then frozen and thawed. Based on these observations, two patients with advanced ANLL in remission underwent marrow ablative therapy followed by autologous reconstitution using TPA-treated bone marrow. Limited normal hematopoiesis was reestablished in both patients, although they subsequently experienced leukemic relapse. These studies demonstrate that in ANLL cells, TPA stimulates growth arrest; in contrast, hematopoiesis is able to proceed both in vitro and in vivo.