Frederick R. Rickles

In situ detection of tissue factor in vascular endothelial cells: correlation with the malignant phenotype of human breast disease.

Expression of tissue factor (TF) in the endothelium has been observed only rarely in human disease and has been thought to be elaborated on the surface of vascular endothelial cells (VECs) in vitro as an artifact of tissue culture. Using monoclonal antibodies and a novel probe for functional TF, we have localized TF to the VECs (and tumor cells) within the tumors of seven patients with invasive breast cancer but not in the VECs (or tumor cells) of benign tumors from ten patients with fibrocystic disease of the breast. The potent procoagulant TF was shown to be a marker of the initiation of angiogenesis in human breast cancer. Further evidence that the TF was the demonstration of a similar distribution of cross–linked fibrin only in the VECs of the malignant tumors. We interpret these data as further support for the concept that tumor cells can activate nearby VECs and regulate blood vessel growth in vivo. Large clinicalpathologic studies will be necessary to determine whether TF is a useful marker for the “switch to the angiogenic phenotype” in human breast disease and/or correlates with the thromboembolic complications of breast cancer.

Response of patients with mild and moderate hemophilia A and von Willebrand's disease to treatment with desmopressin

Desmopressin was administered intravenously to 68 patients with hemophilia and von Willebrands disease of mild or moderate severity to assess the safety, reproducibility, and range of response to this new therapeutic alternative. A rise in factor VIII-von Willebrand factor levels was seen in 64 patients, and the magnitude was sufficient to provide normal hemostasis in 55 to 68 spontaneous or traumatic bleeding episodes, dental procedures, or operations. Thus, our experience shows that most patients with mild or moderate hemophilia and von Willebrands disease can be treated effectively without plasma derivatives. Patients who had two or more infusions of desmopressin at different times had similar responses each time, and members of the same family also had similar responses after desmopressin infusions. Because this drug can be administered without significant side effects, it should have an important role in the management of patients with mild or moderate hemophilia and von Willebrands disease.

Activation of blood coagulation in Crohn's disease. Increased plasma fibrinopeptide A levels and enhanced generation of monocyte tissue factor activity.

We have examined the relationships among activation of blood coagulation, generation of monocyte procoagulant activity, and clinical activity in patients with Crohns disease. Subclinical activation of blood coagulation was measured using a radioimmunoassay for fibrinopeptide A. Fibrinopeptide A levels were strongly correlated with the level of disease activity as measured by the Crohns disease activity index. Patients with active disease who were successfully treated either medically or surgically demonstrated a reduction of fibrinopeptide A levels. Failure of fibrinopeptide A to return to the normal range predicted an early relapse. Monocyte tissue factor generation was assessed in both unstimulated and lipopolysaccharide-stimulated mononuclear cell cultures obtained from the peripheral blood of patients with Crohns disease. A strong correlation (r = 0.89) was observed between plasma fibrinopeptide A levels and monocyte tissue factor generation. These results suggest that monocyte procoagulant generation may contribute to the activation of blood coagulation in this inflammatory bowel disease. Moreover, fibrinopeptide A levels in Crohns disease may provide a useful quantitative measure of inflammatory activity.

Heparin abolishes the chemotherapy-induced increase in plasma fibrinopeptide A levels

PURPOSE, PATIENTS, AND METHODSnBlood coagulation abnormalities are common in patients with cancer, particularly after treatment with chemotherapeutic agents. Chemotherapy has been associated with an increased incidence of thromboembolic events, and patients treated with chemotherapy often develop evidence of local phlebitis, which may lead to loss of venous access. We have utilized the radioimmunoassay for plasma fibrinopeptide A (FPA) and in vitro FPA generation to assess the rate of in vivo blood coagulation and the level of plasma thrombin activity in 16 cancer patients treated with chemotherapy. Eight patients were treated twice, once with chemotherapy alone and once with chemotherapy after an intravenous infusion of heparin (5,000 U).nnnRESULTSnOur results confirm that FPA levels are elevated in most cancer patients. Following chemotherapy, FPA levels were further increased within 45 minutes (mean FPA = 5.2 ng/mL before chemotherapy versus 8.3 ng/mL after chemotherapy, p less than 0.01) and were accompanied by an increase in the FPA generation rate. Infusion of heparin prior to chemotherapy significantly lowered plasma FPA levels and abolished post-chemotherapy FPA generation.nnnCONCLUSIONnThese data suggest that patients receiving chemotherapy express thrombin-like activity in plasma and, therefore, may be at risk for clinically significant intravascular activation of coagulation. Heparin diminished the laboratory evidence of this chemotherapy-related coagulopathy and may have a role in the prevention of thromboembolic disorders in some cancer patients undergoing cytotoxic therapy.

Tissue factor expression in human leukemic cells

Patients with acute leukemia are at increased risk for thrombotic and hemorrhagic complications, particularly those patients with acute promyelocytic leukemia (APL) undergoing induction chemotherapy. These serious complications have been attributed by some authors to the release of tissue factor (TF) procoagulant activity (PCA), particularly during cytotoxic chemotherapy. In previous studies of normal peripheral blood cells, only cells of the monocyte lineage have been found to express TF PCA. Therefore, several questions remain regarding the origin and characterization of the PCA in malignant leukemic cells, particularly those thought to be derived from granulocyte progenitor cells. We utilized a full-length cDNA probe, several monoclonal antibodies (MAbs) and a sensitive one-stage PCA assay to study the expression of TF in the human cell line, HL-60, in human peripheral blood monocytes/macrophages (Mo/Mø) and in highly purified populations of human polymorphonuclear leukocytes (PMN). In the HL-60 cells we detected low but significant levels of TF mRNA and TF antigen (TF:Ag). In unstimulated cells, coordinate increased levels of TF mRNA, TF:Ag and TF PCA expression were noted following phorbol-ester-induced macrophage differentiation of the cells, but a decreased level of TF mRNA with no change in the basal level of TF:Ag expression occurred following retinoic acid-induced granulocyte differentiation of this cell line. Long-term cultures of stimulated mature Mo/Mø demonstrated initial coordinate expression of TF mRNA, TF:Ag and TF PCA, but TF:Ag expression persisted even after 7 days (when TF PCA was undetectable). No TF PCA, TF:Ag or TF mRNA was demonstrated in highly purified populations of human PMN, regardless of culture conditions. Discordant expression of TF mRNA, TF:Ag and TF PCA in HL-60 cells suggests the possibility of novel, post-synthetic mechanisms for the regulation of TF PCA expression, which might be dependent on the phenotypic differentiation level of the cell. Such mechanisms (yet to be defined) might account for the ability of some leukemic cells, which frequently express characteristics of more than one cell line (e.g. monocytes and granulocytes), to express a TF gene product capable of activating blood coagulation.

von Willebrand's disease and hemorrhagic telangiectasia: Association of two complex disorders of hemostasis resulting in life-threatening hemorrhage

The clinical and laboratory findings in a patient with uncontrolled gastrointestinal bleeding secondary to combined hemostatic defects (von Willebrands disease and hemorrhagic telangiectasia) are described. Evidence for von Willebrands disease was found in five family members, but no other affected relative was found to have hemorrhagic telangiectasia. Complete assestivity, factor VIII antigen and von Willebrand factor levels. The patient described also was evaluated for her response to transfusion utilizing these same measurements. Previous reports of the coexistence of hemostatic defects with hereditary hemorrhagic telangiectasia are reviewed. The importance of complete hemostatic evaluation of patients with mucocutaneous bleeding is stressed in light or current knowledge of the diagnostic specificity of available laboratory tests.

The Limulus Blood Cell Secretes α2-Macroglobulin When Activated

Alpha2-macroglobulin, a protease-binding protein that is reactive with almost all endopeptidases, is present in high concentrations in the plasma of the horseshoe crab, Limulus. Alpha2-macroglobulin was demonstrated by its ability to protect the active site of trypsin from inactivation by the macromolecular active site inhibitor, soybean trypsin inhibitor, and by reaction with an antiserum prepared against purified Limulus α2-macroglobulin. The blood cells also contain α2-macroglobulin in a form that is released when washed cells are stimulated to undergo exocytosis by treatment with the ionophore, A23187. Alpha2-macroglobulin is detected in the materials released from the cells during degranulation both by activity in the soybean trypsin inhibitor-protection assay and by immunochemical staining of Western blots. The subunit molecular weight of the cell-associated form of α2-macroglobulin, 185 kDa, is identical to that of the plasma form. The amount of α2-macroglobulin contained within the cells of a given volume of blood is 0.5-2% of the quantity in solution in that volume of plasma. The distilled water lysates of N-ethylmaleimide-stabilized amebocytes used to detect endotoxin (e.g., Limulus amebocyte lysate or LAL) contain relatively large quantities of active α2-macroglobulin. These preparations are essentially free of the principal plasma protein, hemocyanin, indicating that the cells had been well washed prior to lysis.

Research priorities in hereditary hemochromatosis.

The Working Group on Research Priorities, a panel of clinicians, basic scientists, and experts in laboratory medicine and public health, was asked to 1) identify unanswered questions about population screening for hereditary hemochromatosis and other iron overload disorders and 2) recommend specific research studies with which to answer these questions. The Working Group used a formal nominal group technique, described in detail elsewhere [1, 2], to identify and prioritize the specific aims of applied research that would need to be met to provide a scientific basis for population screening. The members of the Working Group generally agreed that hereditary hemochromatosis seemed to be an almost ideal candidate for population screening: It is an inherited disorder that has a high prevalence in the U.S. population; may have serious clinical manifestations leading to premature death; can be identified by safe and reliable screening and diagnostic tests; and, after early diagnosis, can be treated effectively and inexpensively to prevent later complications. In the past two decades, almost all of more than 20 intervention trials [3] have provided evidence of the benefits of screening for iron overload. Practice guidelines for the detection, diagnosis, and management of hereditary hemochromatosis have been published [4]. Genetic testing for the disorder has been made possible by the recent identification of a gene (HFE) that is mutated in most patients with hereditary hemochromatosis [5]. An animal model of hereditary hemochromatosis has been developed in genetically engineered mice that do not express HFE [6]. Despite these favorable factors, the Working Group identified the incomplete characterization of the natural history of hereditary hemochromatosis (that is, the spontaneous evolution of the clinical and laboratory manifestations of iron overload in persons with a defined genotype) as the central remaining problem in determining the value of population screening to the public health. In particular, uncertainty remains about the penetrance of the disease (that is, the proportion of persons with the hemochromatosis genotype who, without treatment, will develop illness as a result of iron overload). Overall, the Working Group agreed that the highest priority for future research on hereditary hemochromatosis should be given to better characterization of the natural history of the condition and to clarification of the relation between genotype (hereditary predisposition) and phenotypic expression (penetrance and severity) of the disease. In addition to noting the need for applied research, the Working Group recognized that we need to develop and evaluate both a national program of laboratory standardization and a broad-based educational program for health care providers, patients, their families, and the general public before we implement a practical program for screening the U.S. population for iron overload. Aims of Applied Research in Hereditary Hemochromatosis Characterization of the Relation between Genotype and Phenotype The most important research aim identified by the Working Group was better characterization of the natural history of the relation between genotype and phenotypic expression in hereditary hemochromatosis and other iron overload disorders with respect to age, sex, ethnicity, and environmental factors. Despite the wealth of information about hereditary hemochromatosis that has already accumulated [3, 4], controlled, population-based studies of the evolution of clinical and laboratory manifestations of iron overload disorders in persons of defined genotype are still considered necessary to provide a scientific basis for the formulation of optimal screening strategies. The available population-based studies of screening for hereditary hemochromatosis all have methodologic limitations that might bias their results toward an overestimation of disease expression [3], and none has included a control group to allow comparison of the frequency of nonspecific symptoms or signs (this is necessary to determine the proportion of these symptoms and signs that may not be due to hereditary hemochromatosis). Studies of families of probands with hereditary hemochromatosis suggest that about 68% of female and 93% of male homozygotes for the HFE mutation meet current clinical diagnostic criteria for the disease [7], but family studies of persons with disease may overestimate disease expression among those with a genetic predisposition. Informed decisions about screening and therapy to prevent the complications of iron overload require accurate information not only about the prevalence and severity of disease manifestations among persons with the hereditary hemochromatosis genotype but also about the relation between iron overload and biochemical measures of iron status (such as transferrin saturation, unsaturated iron-binding capacity, serum ferritin level, and hepatic iron concentration) and the relation of these measures to the development and reversibility of organ damage. More data are needed about the effects on the expression of hereditary hemochromatosis of such factors as age; sex; ethnicity; and environmental factors, including diet, use of nutritional supplements, use of medications, use of alcohol, and viral hepatitis. Identification of a candidate gene (HFE) for hereditary hemochromatosis [5] and development of a DNA-based test for this gene were major advances that now can be used in population-based studies to clarify patterns of clinical expression. It is likely that additional genes for hereditary hemochromatosis will be identified in the future and that different hereditary forms of iron overload are present in different populations. For example, a form of genetically linked primary iron overload that is distinct from hereditary hemochromatosis may be present in persons of African descent [8]. Determination of the Optimal Approach to Screening for Iron Overload The second major research aim identified by the Working Group was to devise an optimal method for screening for hereditary hemochromatosis and other forms of iron overload. Studies should determine the best screening tests and evaluate algorithms for their application. These algorithms could include both phenotypic testing (done by using measures of iron status) and genotypic (DNA-based) testing, alone or in combination; should specify the age range in which the algorithms should be applied; and should include criteria for the identification of affected persons that would provide a suitable balance between adequate rates of detection and acceptable rates of false-positive results. Along with the development of screening algorithms, improvements in laboratory methods (with standardization and quality control for both phenotypic and genotypic tests) were recognized as necessary for the effective implementation of early detection and treatment programs. Analysis of the Cost-Effectiveness of Screening for Iron Overload The third major applied research aim was to conduct a formal analysis of the cost-effectiveness of screening for iron overload. Although numerous studies of cost-effectiveness have been done [3], the cost-effectiveness of screening algorithms could be assessed more realistically if we had a better understanding of the natural history of iron overload disorders (for example, the factors affecting disease expression and the length of time before irreversible organ damage develops). More data are needed before we can decide on the best use of phenotypic and genotypic testing, determine the optimal age for screening, and compare the sensitivities and specificities of various screening procedures. In this analysis, the cost-effectiveness of population screening would be compared with that of screening only affected persons who present for medical attention with signs and symptoms compatible with iron overload or only relatives of persons clinically affected by hereditary hemochromatosis. After the development of a model for population screening, sensitivity analyses could be done to assess the effects of varying levels of prevalence of hereditary hemochromatosis, the performance characteristics of screening and diagnostic tests, the probability of clinical manifestations in affected persons, and the costs of testing and treatment. Results of cost-effectiveness analyses will help clarify the economic implications of a variety of screening strategies. Assessment of the Ethical, Legal, and Social Implications of Screening for Hereditary Hemochromatosis The fourth and final identified research aim was to assess the ethical, legal, and social implications of screening for hereditary hemochromatosis. One of the anticipated impediments to implementation of a screening program was the risk that the genetic information resulting from screening might be used by insurers, employers, or others to deny health care coverage or services to persons identified as being at risk for iron overload. Concern was also raised that a diagnosis of hereditary hemochromatosis could lead to changes in self perception, family interactions, and risk-taking behaviors. Research deemed necessary included studies of 1) ways to ensure privacy and fairness in the use and interpretation of genetic information derived from hereditary hemochromatosis screening, 2) provision and evaluation of counseling for affected persons and their families, 3) methods for obtaining informed consent for screening, and 4) development of programs for effective public and professional education on issues in screening. Studies To Achieve Research Aims The Working Group judged that the four major aims of applied research discussed above could be met with two types of carefully designed studies. Multicenter, Cross-Sectional, Population-Based Study of the Natural History of Hereditary Hemochromatosis The first priority for applied research was the characterization of the natural history of the r

Nε(γ-glutamyl)lysine crosslinks in the blood clot of the horseshoe crab, Limulus polyphemus

Abstract Clots were allowed to form in samples of whole blood taken from the American horseshoe crab, Limulus polyphemus , in the absence and presence of dansylcadaverine (16), and were analyzed for their contents of N e (γ-glutamyl)lysine and γ-glutamyl-dansylcadaverine. Clots obtained without dansylcadaverine yielded significant amounts of N e (γ-glutamyl) lysine product. Clots formed in the presence of dansylcadaverine yielded only γ-glutamyl-dansylcadaverine. Formation of these products reflects on the activity of transglutaminase released from the blood cells during coagulation.

Macrophage Procoagulants, Fibrin Deposition, and the Inflammatory Response

In 1938, Menkin provided a comprehensive view of the inflammatory response: “Inflammation may be broadly defined as the complex vascular, lymphatic, and local tissue reaction elicited in higher animals by the presence of microorganisms or of nonviable irritants. It represents a basic reaction to injury, in which the deleterious agent tends to be localized and ultimately destroyed.” Menkin characterized several phases in the development of the inflammatory lesion beginning with increased fluid passage through the capillary endothelial wall and the formation of a fibrinous network which immobilizes the irritant. The lesion is then completed by the development of local thrombi, occlusion of draining lymphatics, and subsequent infiltration of leukocytes. Menkin proposed that the early fixation of the irritant allowed an interval during which leukocytes could assemble for the purpose of phagocytosis. Furthermore, these three phases—local fluid influx, fixation in a focal fibrinous meshwork, and progressive leukocyte infiltration—represent a necessary prelude to the development of immunity.