Heparin-binding proteins are involved in thrombin time variability in normal and patient plasmas.

The ability of plasma proteins to neutralize the anticoagulant activity of heparin was studied using a thrombin time assay with 20 normal adult and 207 patient plasma samples. The thrombin times of normal adult plasmas spiked with the same amount of heparin were found to vary significantly, and this variability was attributed to differences in heparin-binding proteins among individuals. This possibility was investigated by determining thrombin times for a variety of plasma samples following proteolytic digestion. A study of patient pooled plasma showed that incubation with a protease increased the thrombin time from 23 s to > 300 s, suggesting that the anticoagulant activity of heparin could be neutralized by plasma proteins and released with proteolytic digestion. Incubation of the digested plasma with heparinase I returned the thrombin time almost to control values. In addition, patients who received prior heparin injections had different responses to similar dosages of heparin, as indicated by differences in thrombin times. Thus, the anticoagulant activity of heparin may be affected by the balance between free heparin and protein-bound heparin in plasma.

Antibodies to endothelial cells identify myocardial damage and predict development of coronary artery disease in patients with transplanted hearts.

BACKGROUND: Transplant-induced coronary artery disease is a leading cause of graft failure in cardiac allograft recipients after the first year of transplantation, but there presently is no test to identify patients at high risk for developing the disease. Our research is focused on development of a predictive test to identify patients at high risk of developing the disease. METHODS: Sixty-eight cardiac allograft recipients transplanted and followed at Methodist Hospital between 1982 and 1996 were studied. Serial annual angiograms were used to diagnose coronary artery disease, and serial endomyocardial biopsies were used to detect cellular infiltrates and microvascular disease. Biopsy-matched serum samples were used for cardiac troponin-T determinations as measures of myocardial damage, and serum antibodies to endothelial cells were determined by using flow cytometry, enzyme-linked immunosorbent assay and immunoblotting techniques. The endothelial antibody data were evaluated statistically for associations with angiographic changes, biopsy findings and biochemical evidence of myocardial damage. FINDINGS: Antibodies to endothelial cells were identified by all three techniques, and significant associations were found for the amount of antibody identified by Western immunoblotting with histological rejection grades in biopsies, which were confirmed immunocytochemically as macrophages (p<0.01) and T lymphocytes (P = 0.03). These antibodies also associated significantly with vascular antithrombin depletion (p = 0.02), biochemical evidence of myocardial damage (p = 0.005) and subsequent development of coronary artery disease (p = 0.03). INTERPRETATION: The significant association of anti-endothelial antibodies with cellular infiltrates, depletion of vascular antithrombin and myocardial damage suggests a role for antibody in the development of transplant-induced arteriopathy. The significant association of antiendothelial antibodies with the future development of coronary artery disease further suggests that assessment of these antibodies may provide a non-invasive test to predict the development of transplant-induced coronary artery disease.

Qualitative and quantitative studies of autoantibodies to phospholipids in diabetes mellitus.

Diabetes mellitus is associated with vascular and neurological complications. We have investigated the presence of antibodies to phospholipids and to phospholipid binding plasma proteins in blood samples collected from 68 clinically and biochemically characterized type I and type II diabetic patients and from 252 healthy blood donor controls. Each sample was analysed for antibodies to three phospholipids (cardiolipin, phosphatidylserine and phosphatidylethanolamine), the antibody isotypes (IgA, IgG and IgM), and whether antibody activity was plasma protein-dependent. Patients were considered to have anti-phospholipid antibodies when one or more of these 18 tests was found above predetermined control values. The results of these experiments revealed an increased incidence of anti-phospholipid antibodies in diabetic patients compared with control subjects. The incidence of IgA isotype to phosphatidylethanolamine was higher than the incidence of other isotypes to other phospholipids, and their reactivities were independent of phospholipid-associated proteins. In addition, these antibody findings were studied for associations with prothrombin degradation products, activated factor VII and activated protein C, and with the incidence of diabetic complications. The anti-phosphatidylethanolamine antibody association with proliferative retinopathy was significant.

Unique epitopes of lactoferrin expressed in human cytotrophoblasts involved in immunologic reactions.

OBJECTIVE: Lactoferrin is an iron-binding protein that has been implicated in protection against infections and allogeneic recognition reactions and in the control of cell growth. We studied the biochemical characteristics and expression of the unique lactoferrin epitopes (LF(1)) in human placentas. STUDY DESIGN: Immunohistologic studies of normal human term placentas were done by using monoclonal antibodies to LF(1). Double-antibody experiments were done by using monoclonal antibodies to markers of inflammation (macrophages, human leukocyte antigen [HLA-DR]). LF(1) was studied immunochemically by using lactoferrin fragments produced by the reaction of lactoferrin with trypsin or N-glycanase. RESULTS: Anti-LF(1) monoclonal antibodies reacted with most interstitial cytotrophoblasts in the basal plate and with villous cytotrophoblasts of some but not all chorionic villi. Cytotrophoblasts expressing LF(1) were associated with large numbers of HLA-DR-reactive macrophages. Anti-LF(1) monoclonal antibodies reacted with 2 distinct tryptic fragments of lactoferrin, and these reactivities were not affected by treatment with N-glycanase. CONCLUSION: Placental cytotrophoblasts express unique epitopes of lactoferrin (LF(1)). Such expression is increased in the presence of activated macrophages. This expression could be an extraembryonic response to inflammation and maternal allogeneic recognition as an effort to protect trophoblastic cells. The LF(1) epitopes might represent conserved polypeptide epitopes on 2 homologous lobes of lactoferrin.

Localization and characterization of antithrombin in human kidneys.

Antithrombin is a serine protease inhibitor that is critical in maintaining a thromboresistant vasculature. The association between low serum antithrombin concentration and renal disease suggests that the kidney plays a role in the conservation of plasma antithrombin. We used immunohistochemical techniques to determine the spatial distribution, heparin binding characteristics, and intracellular and intercellular localization of antithrombin in biopsy specimens (n = 53) of human donor kidneys obtained at the time of transplantation. In the renal cortex, double antibody techniques demonstrated the presence of intracellular antithrombin in proximal tubule epithelial cells. The reactivity was granular and was co-localized with vesicle-like structures. Distal and collecting tubules did not demonstrate intraepithelial antithrombin reactivity. No tubule structures in the medullary region demonstrated intracellular antithrombin, but all these structures showed intense basement membrane antithrombin reactivity. Double antibody techniques also demonstrated that the heparin binding domain of intraepithelial antithrombin was occupied. Semiquantitative scores for intraepithelial antithrombin were significantly decreased in renal biopsy specimens obtained 30 min after anastomosis compared with biopsies from the same organ obtained before anastomosis. These findings suggest that antithrombin, probably in association with heparin or heparan sulfate, is internalized by renal proximal epithelial cells. Although the ultimate fate of intraepithelial antithrombin is not known, this may represent a mechanism by which the kidney helps to maintain plasma antithrombin concentrations.

Protein-bound heparin/heparan sulfates in human adult and umbilical cord plasma.

We used thrombin times and a competitive radiometric assay to identify, quantitate and characterize endogenous heparin-like molecules in umbilical cord (n = 58) and normal adult (n = 25) plasma. Thrombin times for cord plasma (29.6+/-3.6 s) were significantly longer (p< or = 0.0005) than those for adult plasma (18. 9+/-2.3 s), suggesting increased endogenous heparins. A radiometric assay based on the displacement of (125)I-heparin from protamine-Sepharose revealed that protease-digested plasma contained heparin/heparan sulfate, and plasma that was not digested with protease appeared not to contain heparin/heparan sulfate. More heparin/heparan sulfate was identified in cord than in adult plasma (p< or =0.05), but heparinase digestion produced significantly (p< or =0.001) reduced concentrations of heparin/heparan sulfate in only 39% of the samples. The lack of heparinase sensitivity in 61% of the protease-digested samples apparently was due to low molecular weight (LMW) heparins, for control heparin fragments of 5 kD that did not extend thrombin times were also less affected by heparinase, but the same LMW heparins were detected by radiometric assay. Despite normal thrombin times in all samples, the amounts of endogenous heparin/heparan sulfate identified in protease-digested samples by radiometric assay were of sufficient concentrations to produce inordinately prolonged thrombin times when compared with the same concentrations of unfractionated heparin. Collectively, these findings suggest the presence of a plasma reservoir of endogenous heparin/heparan sulfates in normal cord and adult plasma. These endogenous heparin/heparan sulfates are bound to plasma proteins, and an as yet undetermined proportion of these bound heparin/heparans are most likely LMW molecules.

Serum cardiac troponin-T concentrations predict development of coronary artery disease in heart transplant patients.

BACKGROUND: Development of coronary artery disease in cardiac allograft recipients is the major cause of graft failure after the first year of transplantation. Unfortunately, there is no noninvasive method of identifying patients at greatest risk of developing this disease. We have asked whether serum concentrations of cardiac troponin-T predict development of coronary artery disease. METHODS: Annual coronary angiograms, serial endomyocardial biopsies, and serum cardiac troponin-T concentrations were obtained from 68 cardiac transplant patients who were followed for 68.8+/-11.9 months after surgery. Troponin-T concentrations were measured by using an enzyme-linked immunosorbent assay, and biopsies were assessed histologically for rejection grades and immunohistochemically for cellular infiltrates, arteriolar endothelial activation, fibrin deposits, and vascular fibrinolytic and anticoagulant components. RESULTS: Troponin-T values did not associate with demographic, clinical, or laboratory findings, but they significantly associated with arteriolar endothelial activation (P<0.001), fibrin deposition (P<0.001), depletion of vascular fibrinolytic (P=0.007) and anticoagulant components (P=0.02), and infiltration of macrophages (P <0.001) but not T lymphocytes (P=0.36). Troponin-T concentrations also significantly associated with future development of coronary artery disease (P<0.001). Patients with persistent troponin-T values of 0.10 ng/ml or greater were found to develop the disease within 8.7+/-2.1 months, whereas patients who had initial troponin-T values of 0.10 ng/ml or greater and subsequently fell and remained below 0.10 ng/ml did not develop coronary artery disease in 40 months. CONCLUSIONS: Troponin-T concentrations significantly associated with macrophage infiltrates, microvascular fibrin deposits, arteriolar endothelial activation, depletion of vascular fibrinolytic and anticoagulant components, and the future development of coronary artery disease. The troponin-T assay is an outpatient procedure performed on small amounts of blood at little cost, risk, or inconvenience, and it appears to be the first biochemical predictor of transplant-induced coronary artery disease.

The pre-conditioning incidence of antiphospholipid antibodies is not significantly increased in patients with bone marrow transplant-related organ dysfunction.

Hepatic dysfunction resulting from hepatic veno-occlusive disease (VOD) is a common complication of bone marrow transplantation (BMT). Some investigators believe that hepatic dysfunction, along with pulmonary and central nervous system (CNS) dysfunction, is part of a systemic disorder called multiple organ dysfunction syndrome (MODS). Endothelial damage by pretransplant chemo-radiation and activation of hemostasis are considered early events in the development of hepatic VOD. The pathological mechanism leading to fibrous obliteration of hepatic vessels may also take place in pulmonary and CNS vessels. Since antiphospholipid antibodies (aPA) are associated with venous and arterial thrombosis, which can lead to vessel occlusion, we asked if the incidence of aPA before conditioning was greater in patients who developed MODS following BMT. Samples drawn before pretransplant chemo-radiation from 57 patients who subsequently developed MODS and 55 control patients who did not develop MODS were studied blindly for aPA by ELISA. The number of aPA-positive patients who developed MODS (10/57), compared to the number of aPA-positive patient controls who did not develop MODS (7/55) was not statistically significant (P = 0.48). Our data indicate that the incidence of aPA before conditioning was not greater in patients who developed MODS, including hepatic VOD, following BMT.

Tubular antithrombin at transplantation determines subsequent renal allograft function.

BACKGROUND: Antithrombin is found in the microvasculature and tubules of normal and transplanted human kidneys. Although depletion of vascular antithrombin is associated with renal allograft dysfunction, neither the distribution nor clinical significance of tubular antithrombin is known. METHODS: Changes in tubular antithrombin in biopsy specimens (n=41) obtained from donor kidneys at transplantation were studied immunohistochemically. The relationship between these changes and subsequent graft function was analyzed. RESULTS: Granular intracellular antithrombin was found only within the proximal tubular epithelium. Allografts with depleted tubular antithrombin at transplantation (n=20) had significantly greater plasma creatinine concentrations at posttransplant days 3 (P < 0.001) and 5 (P < 0.03) than allografts with normal tubular antithrombin (n=21). Indeed, depletion of tubular antithrombin at transplantation correlated with the degree of graft dysfunction at 3 days after transplantation. CONCLUSIONS: Depleted tubular antithrombin at transplantation is associated with reduced early graft function, and this may identify patients at risk of a complicated follow-up.

Heparin regulates ICAM-1 expression in human endothelial cells: an example of non-cytokine-mediated endothelial activation.

Activated endothelial cells up-regulate the expression of several molecules on their plasma membranes, including intercellular adhesion molecule-1 (ICAM-1). The role of heparin in regulating endothelial cell gene expression is unclear. We thus have investigated the ability of heparin to regulate ICAM- gene expression by using flow cytometry and the ribonuclease protection assay with human umbilical vein and aortic endothelial cells cultured in growth medium supplemented with 90 [microg/ml heparin (heparin-sufficient, HS) or in growth medium without added heparin (heparin-deficient, HD). We found that HD medium increased plasma membrane protein and mRNA for ICAM-1 but not for HLA-DR, even though both ICAM-1 and HLA-DR protein and mRNA were inducible by gamma interferon (IFN-gamma). In addition, phorbol ester and IFN-gamma increased the expression of plasma membrane ICAM-1 or ICAM-1 and HLA-DR, respectively, more in HD medium than in HS medium. We found that the HD-mediated increase of ICAM- mRNA was reversible by the addition of heparin, and that the half-life of ICAM-1 mRNA was the same in both HS- and HD-treated cells. Also, heparin was found to suppress increases in ICAM-1 mRNA at a concentration as low as 5 microg/ml. These findings indicate that heparin deficiency induces endothelial activation characterized by increased ICAM-1, and that such induction is not dependent on cytokines or endotoxin. The modulation of ICAM-1 expression by heparin appears to occur at the transcriptional level. Thus, heparin may have a role in regulating endothelial function by affecting the expression of ICAM-1, thereby impacting upon the trans-endothelial trafficking of leukocytes.

Myocardial fibrin deposits in the first month after transplantation predict subsequent coronary artery disease and graft failure in cardiac allograft recipients.

PURPOSE: To determine whether fibrin deposition during the first month following cardiac transplantation predicts development of coronary artery disease and graft failure in cardiac allograft recipients. PATIENTS AND METHODS: We prospectively studied 121 consecutive adult patients who received cardiac transplants between 1988 and 1995. Serial endomyocardial biopsies obtained during the first month posttransplant (2.3 + 0.6 biopsies/patient) were studied immunohistochemically for fibrin deposits. Patients were followed up with annual angiograms (3.2 + 1.7/patient) evaluated with side-by-side comparisons for the presence and progression of coronary artery disease. RESULTS: All pretransplant biopsies were fibrin-negative; 60 allografts (50%) remained without fibrin, and 61 (50%) contained fibrin during the first posttransplant month. Of allografts with fibrin, 72% developed coronary artery disease, while 27% of allografts without fibrin developed the disease (P <0.001). Coronary artery disease was progressive in 61% of allografts with fibrin, and in 25% of allografts without fibrin (P <0.001). Graft failure was more frequent and time-to-graft-failure occurred earlier in patients whose allografts had fibrin during the first month after transplantation (P <0.001). CONCLUSIONS: Fibrin in biopsies during the first month after transplantation identifies patients at high risk for developing coronary artery disease or graft failure, thereby allowing the opportunity to initiate preventive procedures.

Endothelial activation and development of coronary artery disease in transplanted human hearts.

CONTEXT: The development of coronary artery disease in heart transplants is often associated with graft failure. Early detection of allografts prone to develop this disease is essential to institute new therapeutic approaches that could prolong allograft function. OBJECTIVE: To determine if early activation of arterial/arteriolar endothelium predicts the development of coronary artery disease, graft failure, or both in transplanted human hearts. DESIGN: Prospective cohort study. SETTING: Heart Transplant Center. PARTICIPANTS: A total of 121 consecutive adult cardiac allograft recipients who received transplants between 1988 and 1995 and were followed up through 1996. MAIN OUTCOME MEASURES: Development of coronary artery disease and graft failure. METHODS: Immunocytochemistry was performed on serial endomyocardial biopsy specimens to evaluate endothelial activation markers (intercellular adhesion molecule-1 and histocompatibility antigen HLA-DR) in arteries and arterioles. The presence and progression of coronary artery disease was evaluated by annual coronary angiograms with side-by-side comparisons. RESULTS: None of the 121 donor hearts showed arterial/arteriolar endothelial activation before transplantation. Arterial/arteriolar endothelial activation was present in 78 and absent in 43 of 121 allografts during the first 3 months after transplantation. The time of appearance and the proportion of biopsy specimens showing endothelial activation during these first 3 months were significantly associated with the risk of developing coronary artery disease, the progression of the disease, and the time required to develop the disease (P<.001). Significantly more patients with arterial/arteriolar endothelial activation died or received a second transplant (P<.001). CONCLUSIONS: Activation of arterial/arteriolar endothelium in transplanted human hearts predicts development of coronary artery disease and increased risk of graft failure.

Is the drug-responsive NADH oxidase of the cancer cell plasma membrane a molecular target for adriamycin?

Enhanced growth inhibition and antitumor responses to adriamycin have been observed repeatedly from several laboratories using impermeant forms of adriamycin where entry into the cell was greatly reduced or prevented. Our laboratory has described an NADH oxidase activity at the external surface of plasma membrane vesicles from tumor cells where inhibition by an antitumor sulfonylurea, N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl)urea (LY181984), and by the vanilloid, capsaicin (8-methyl-N-vanillyl-6-noneamide) correlated with inhibition of growth. Here we report that the oxidation of NADH by isolated plasma membrane vesicles was inhibited, as well, by adriamycin. An external site of inhibition was indicated from studies where impermeant adriamycin conjugates were used. The EC50 for inhibition of the oxidase of rat hepatoma plasma membranes by adriamycin was several orders of magnitude less than that for rat liver. Adriamycin cross-linked to diferric transferrin and other impermeant supports also was effective in inhibition of NADH oxidation by isolated plasma membrane vesicles and in inhibition of growth of cultured cells. The findings suggest the NADH oxidase of the plasma membrane as a growth-related adriamycin target at the surface of cancer cells responsive to adriamycin. Whereas DNA intercalation remains clearly one of the principal bases for the cytotoxic action of free adriamycin, this second site, possibly related to a more specific antitumor action, may be helpful in understanding the enhanced efficacy reported previously for immobilized adriamycin forms compared to free adriamycin.

Zinc as a cofactor for heparin neutralization by histidine-rich glycoprotein.

We have studied the ability of histidine-rich glycoprotein (HRG) to neutralize the anticoagulant activity of heparin in plasma and in a purified component clotting assay. Addition of HRG to plasma or to the purified component assay did not neutralize the anticoagulant activity of heparin unless micromolar concentrations of zinc were present. Higher zinc concentrations were required for citrated than for heparinized plasmas due to competition of citrate with HRG for zinc binding. Zinc concentrations as low as 1.25 microM revealed HRG to be a powerful competitor of antithrombin for heparin in the purified component assays. HRG binding of heparin also was shown by affinity chromatography of HRG from immobilized heparin in the presence and absence of zinc. In the absence of zinc, HRG was eluted by 0.1 M NaCl, but, in the presence of zinc, elution of HRG required 1.0 M NaCl. Investigation of other divalent cations (copper and magnesium) indicated that augmentation of heparin binding by HRG in the presence of antithrombin was restricted to zinc. The HRG.Zn complex effectively competes with antithrombin for heparin, which restricts the availability of heparin to bind antithrombin and allows thrombin-mediated fibrinogenesis to proceed unimpeded. This could be initiated by zinc released from activated platelets.

Vascular endothelial growth factor expression in transplanted human hearts.

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen thought to play an important role in coronary collateral vessel formation. We used immunocytochemistry to determine VEGF expression in biopsies (n = 283) of transplanted human hearts (n = 109) with and without microvascular fibrin. Measures of vascular fibrin, alpha 2 plasmin-inhibitor (a2Pl), macrophages, neutrophils, and serum cardiac troponin T titers were used to evaluate myocardial damage. Antibody to T lymphocytes was used to evaluate cellular rejection, and HLA-DR, ICAM-1, and PAL-E antibodies were used to assess endothelial cell activation and phenotypic changes in the microcirculation. No VEGF immunoreactivity was detected in control donor hearts without fibrin, but the proportion of biopsies demonstrating VEGF immunoreactivity increased significantly in allografts with increasing fibrin and a2PI reactivity (P = 0.0001). VEGF immunoreactivity was confined to areas of fibrin deposition and was associated with infiltrates of macrophages and neutrophils (P < 0.0001), but not with T cells (P = 0.10). Biopsies with fibrin/VEGF reactivity were associated with increased capillary endothelial cell HLA-DR, ICAM-1, and PAL-E reactivity. In a subset of patients, serum cardiac troponin-T values were greater in patients with VEGF-positive (n = 21) than VEGF-negative (n = 19) biopsies (P = 0.05). Nested RT-PCR demonstrated that biopsies with and without fibrin/VEGF immunoreactivities expressed VEGF121, VEGF165, and VEGF189 variants, with VEGF165 being the dominate variant. These results indicate that endogenous VEGF is expressed locally following vascular thrombosis and myocardial cell damage, and that VEGF expression may be related to endothelial cell activation and phenotypic changes found in the microcirculation of cardiac allografts.

Vascular tissue plasminogen activator and the development of coronary artery disease in heart-transplant recipients.

BACKGROUND: An aggressive and potentially fatal form of coronary artery disease may develop after cardiac transplantation. We studied the role of vascular tissue plasminogen activator (t-PA), the primary mediator of fibrinolysis, in the development of this problem. METHODS: We studied 78 consecutive recipients of cardiac allografts over a five-year period, and we collected follow-up data over a mean (+/- SE) of 32.5 +/- 2.0 months. The patients were studied with ventricular function tests, serial endomyocardial biopsies (16.6 +/- 0.5 per patient), and annual coronary angiography. Measurements of t-PA and its inhibitor were performed immunocytochemically on unfixed cryostat sections of endomyocardial-biopsy specimens with the use of monoclonal antibodies to t-PA and its inhibitor. RESULTS: In biopsy specimens obtained during the first three months of follow-up, 38 allografts had a normal distribution of t-PA in arteriolar smooth-muscle cells, whereas in 40 allografts there was depletion of t-PA that persisted in subsequent follow-up. Coronary artery disease developed during follow-up in 31 of 40 allografts (78 percent) with depletion of t-PA, but the disease developed in only 9 of the 38 allografts (24 percent) with normal t-PA levels (P < 0.001). Allografts with depletion of t-PA also had the t-PA inhibitor and were at greater risk for earlier and more severe disease than were allografts with normal arteriolar t-PA levels. Twelve patients whose allografts were depleted of t-PA either received a second transplant or died, whereas only one of the patients whose allografts had persistently normal t-PA levels died (P < 0.001). CONCLUSIONS: These findings reveal an association between the depletion of t-PA from arteriolar smooth-muscle cells and the subsequent development of coronary artery disease and decreased graft survival. Although we cannot be certain about a cause-and-effect relation, our data suggest a possible role for deficient fibrinolysis in the development of coronary artery disease in transplanted human hearts.

Heparan-dependent endothelial antithrombin binding is increased by butyrate.

Heparin biosynthesis involves a critical early step of N-deacetylation which is inhibited by the short chain fatty acid n-butyrate. Such inhibition causes mast cells to produce heparins with high affinity for antithrombin (AT). We have cultured endothelial cells in media supplemented with short chain fatty acids and have found that isobutyric, propionic and valeric acids cause significant increases in endothelial binding of AT measured by flow cytometry, but n-butyric acid was the most effective fatty acid to increase AT binding. Such binding,was heparan sulfate-dependent, for it was decreased significantly by pre-treatment of the cells with heparinase. These findings suggest that inhibition of N-deacetylation in heparan biosynthesis affects sulfation and results in the distribution of negative charges and conformation changes within the heparan domain that binds AT to endothelial plasma membranes. These changes also were associated with up-regulation of the intercellular adhesion molecule-1, which is a marker of endothelial activation.