Identification and characterization of an endothelial, cell-specific antigen with a monoclonal antibody.

The purpose of these studies was to use monoclonal antibodies to identify and characterize plasma membrane components unique to the vascular endothelium. Our assumption is that such components may perform some of the specialized functions of the endothelium and, by their identification with antibody probes, we may be able to study further their function and structure. Thus, primary cultures of human umbilical vein endothelium were used to immunize mice whose spleen cells were fused with the mouse myeloma cell NS-1. HEC-1 is a monoclonal antibody derived from such a fusion that appears to react with an antigen located only on endothelial cells. The antigen has been characterized by immunoprecipitation and polyacrylamide gel electrophoresis as a glycoprotein with a mol wt of 180,000 daltons under nonreducing conditions and 90,000 daltons under reducing conditions. Despite a close resemblance to a membrane component shown by others to be a receptor for transferrin, several lines of evidence reported in this paper indicate that this is not the function of the HEC-1 antigen. These data show that monoclonal antibodies can be used to identify and characterize membrane components of the vascular endothelium. Moreover, these probes can be used to inquire about the structure and function of the antigen with which they react.

Catechol estrogens and thrombosis: differential effect of 2-hydroxyestradiol and estradiol on prostacyclin release.

High estrogen burden states, such as that seen in women who ingest oral contraceptive preparations, are associated with increased thrombosis. In other instances, however, estrogens decrease thrombogenicity. Variations in conversion to active metabolites could explain this paradox. Thus, we examined the influence of estradiol and a major metabolite, 2-hydroxyestradiol (catechol estrogen), on prostacyclin release from cultured human umbilical venous endothelium. In micromolar concentrations, both compounds inhibited prostacyclin (PGI2) release from bradykinin-, arachidonic acid-, and ionophore-stimulated, as well as unstimulated monolayers. However, when thrombin was used to stimulate release, estradiol enhanced, while 2-hydroxyestradiol inhibited, PGI2 formation. These sex steroids probably exert their influence at micromolar concentrations through a non-specific interaction with the cell membrane, and may be of importance in the predisposition to thrombosis suffered by those who use oral contraceptives.

PIP: High estrogen burden states, such as those seen in women who ingest oral contraceptive (OC) preparations are associated with increased thrombosis. In other instances, however, estrogens decrease thrombogenicity. Variations in conversion to active metabolites could explain this paradox. Thus, we examined the influence of estradiol and a major metabolite, 2-hydroxyestradiol (catechol estrogen), on prostacyclin release from cultured human umbilical venous endothelium. In micromolar concentrations, both compounds inhibited prostacyclin (PGI2) release from bradykinin-, arachidonic acid-, and ionophore-stimulated, as well as unstimulated monolayers. However, when thrombin was used to stimulate release, estradiol enhanced, while 2-hydroxyestradiol inhibited, PGI2 formation. These sex steroids probably exert their influence at micromolar concentrations through a nonspecific interaction with the cell membrane, and may be of importance in the predisposition to thrombosis suffered by those who use OCs.

Role of the vascular endothelium.

A possible explanation for the non-thrombogenic effect of the endothelium is the presence of prostacyclin, (PGI2), the potent inhibitor of platelet aggregation and adherence, which is produced and released by the endothelium in response to various stimuli. Removal of PGI2 from the endothelium did not increase baseline platelet adherence, but did increase thrombin-induced platelet adherence from 4 to 60%. Additions of exogenous PGI2 at low concentrations reversed the enhanced thrombin-induced platelet adherence under these conditions. Although it is unlikely that prostacyclin is the sole factor regulating platelet adherence to the endothelium, it appears to play a major role in the interaction of platelets with components of the blood vessel wall. Conditions which predispose to adherence of platelets to the vessel wall may involve entrapment of tumor cells and lead to metastasis formation. Whether prostacyclin and other factors involved in the non-thrombogenic character of the vascular endothelium provide a significant defense against attachment of tumor cells is not known, but the potential for such a primary or secondary role clearly exists. In our studies, prostacyclin did not appear to influence the adherence of Raji lymphoma cells to the endothelium. Additional studies are indicated to correlate adherence of tumor cells to the vascular wall with their potential for formation of metastatic lesions.

Comparative effect of ibuprofen on endothelial and platelet prostaglandin synthesis.

Ibuprofen (Motrin) decreases infarct size in animal models of myocardial infarction. Inasmuch as this effect might be related to the inhibitory effect of ibuprofen on platelet function, we have evaluated its effect on platelet and endothelial prostaglandin synthesis. Human umbilical vein endothelial cultures and washed human platelets were studied. Thromboxane (TX)A2 was determined by radioimmunoassay for its stable and product TXB2 and prostacyclin was determined by radioimmunoassay for its stable end product 6-keto-prostaglandin F1 alpha. At all concentrations of ibuprofen tested, platelet TXA2 and endothelial prostacyclin synthesis were inhibited to a similar degree. Unlike aspirin, the effect of ibuprofen on prostaglandin synthesis was rapidly reversible in both endothelial and platelet systems after removal of the drug. Ibuprofen was also shown to interfere with the irreversible inhibitory effect of aspirin, suggesting that ibuprofen and aspirin might compete for binding by the cyclooxygenase. Inhibition of endothelial and platelet prostaglandin synthesis was nearly complete with 100 microM concentrations of aspirin (100%) and ibuprofen (99%). Thrombin-stimulated platelet adherence to endothelial monolayers was related inversely to the amount of prostacyclin produced by the endothelium. Adherence was maximal at concentrations (100 microM) of ibuprofen and aspirin producing near complete inhibition of prostaglandin synthesis. Maximal adherence with both drugs was comparable (28 vs. 31%). Ibuprofen showed no preferential inhibitory effect on TXA2 synthesis and, at a 100 microM concentration, was as effective as aspirin in the systems studied.

The safety of weekly plateletpheresis: effect on the donors' lymphocyte population.

Ten normal donors were monitored before, during, and after ten weekly donations of platelet concentrates prepared using the Haemonetics Model 30 blood processor. Analysis of these data indicates about a 20 per cent decrease of lymphocytes, with B cells being significantly decreased in half of these patients. While coincident immunoglobulin deficiencies did not develop in this short-term study, it is presumed that these would occur with a prolongation of platelet donations, as has been noted in previous studies. Additional studies are indicated to further delineate the lymphocyte depletion in normal platelet donors.