The patterns of oxidative coagulative injury described in this article were observed in extensive studies of blood morphology in a host of acute and chronic cardiovascular as well as non-cardiovascular disorders, including advanced IHD, unstable angina, congestive heart failure, cardiac arrhythmias, hypertensive crises, acute and chronic viral and bacterial infections, fungemia, acute and chronic atopic disorders, chemical sensitivity reactions, acute and chronic degenerative disorders and malignant diseases.

Erythrocyte Membrane Damage and Lysis in AA Oxidopathy
Erythrocytes, when observed with an ordinary bright-light microscope in stained smears of peripheral blood, appear as rigid, biconcave, disc-shaped corpuscles. When examined with a high-resolution, phase-contrast microscope in freshly prepared unstained smears, these cells are seen as pliable, round cells that readily change their shape to ovoid, triangular, dumbbell, or irregular outlines to squeeze past other erythrocytes in densely populated fields. Such cells resume their regular rounded contour as soon as they find open space.
 
Erythrocytes may be expected to show evidence of oxidative damage earlier than other blood corpuscles since these cells transport oxygen, the most important oxidizer in the body. Furthermore, unlike the leukocyte cell membrane which is sturdy and uniquely equipped with enzymatic antioxidant defenses against oxidative stresses of microbial invaders, the erythrocyte membrane is more permeable (to facilitate oxygen uptake and delivery) and, hence, may be deemed more vulnerable. Our microscopic findings provide some evidence for such theoretical considerations. The earliest and most common abnormalities we observed in AA oxidopathy are erythrocyte membrane irregularities and cell deformities. As oxidopathy progresses, an increasing number of red cells show morphologic abnormalities and some cells appear as ghost outlines. Many erythrocytes show surface wrinkling, teardrop deformity, sharp angulations and spike formations. Other changes include rouleaux formations and zones of plasma congealing around damaged erythrocytes.. Some zones of plasma congealing sometimes appear to form spontaneously (without a discernable cause) in close vicinity of damaged erythrocytes and leukocytes.

We established the oxidative nature of plasma and cellular abnormalities described above by demonstrating their reversibility with antioxidants such as vitamin E, taurine, vitamin A, and vitamin C, reported previously¹² but not shown here. Parenthetically, we add that we have observed similar evidence of erythrocyte membrane injury in diverse clinical entities associated with accelerated molecular injury such as disabling chronic fatigue, fibromyalgia and a host of severe nutritional, ecologic and autoimmune disorders.

Erythrocyte Homogenate, Free Iron and AA Oxidopathy
 In deliberations of atherogenesis, the issues of oxidative injury to erythrocytes and the presence in the plasma of free hemoglobin leached from damaged red cells—and the presence of excess iron in the plasma as a result of those factors—are seldom, if ever, addressed. Our morphologic findings lead us to propose that oxidative erythrocyte injury plays an important role in the genesis of AA oxidopathy and, hence, atherogenesis. We observed erythrocyte membrane damage and lysis with high frequency in many acute ischemic coronary syndromes and, less often, in patients with advanced IHD but without severe, acute coronary ischemia.
 
Iron, like oxygen, is a molecular Dr. Jekyll and Mr. Hyde. It is needed for molecular transport (in hemoglobin for oxygen), for storage (in myoglobin), for energy functions (in cytochrome oxidase and other cytochromes), for respiration (in non-heme-iron proteins), and for antioxidant defenses (in catalase). In its Mr. Hyde role, iron (in free form) is a potent oxidant and catalyzes the generation of many dangerous oxygen-derived radicals.^175-182 In health, the Mr. Hyde roles of iron are minimized by transferrin, an iron-binding protein that rigidly limits the availability of free iron. In normal plasma, only 20 to 30 percent of transferrin occurs in a saturated state.
 
Free hemoglobin has been considered a dangerous protein—a biological Fenton catalyst.¹⁷⁹ It rapidly quenches free radicals in a highly oxidizing environment and becomes oxidized, thus turning into a potent oxidant. It is readily degraded by H2O2 to release free iron, which initiates and propagates several free radical reactions.^182-183 Hemoglobin reacts with H2O2 to produce a protein-bound oxidizing species capable of causing lipid peroxidation.¹⁸⁴ Free hemoglobin also avidly binds with nitric oxide radicals and induces vasospasm, triggering yet other oxidizing events, which, in turn, feed the "oxidative fires" of AA oxidopathy.
 
Beyond ample evidence of the destructive oxidizing capacity of erythrocyte-derived factors discussed above, there is also direct evidence that red blood cells play a role in atherogenesis. Sambrano et al.¹⁸⁵ and colleagues have shown that certain receptors on macrophages for oxidized LDL also bind to oxidatively-injured red cells prior to their internalization and lysis. Oxidatively-modified lipid, proteins, and carbohydrate moieties of erythrocyte membranes can be expected to play a host of roles in oxidative coagulopathy and AA oxidopathy, just as they do in attachment, endocytosis, membrane fusion, and viral hemagglutination in viral infections.^186-189 We may point out in this context, as shown by Oda et al.¹⁸⁹ that oxyradicals play the key pathogenetic roles in virus-induced illness. As we discuss in Part II of this article, a growing body of evidence points to the roles of strong inflammatory, infectious and autoimmune mechanisms in atherogenesis. It seems obvious to us that additional evidence for inflammatory and immunogenic roles of erythrocyte-derived factors in oxidative coagulopathy, AA oxidopathy, atherogenesis and IHD will be forthcoming as those areas are explored further in the future. Of considerable interest in this context is the matter of electrostatic interactions among oxidatively damaged erythrocyte membranes and other oxidized elements in the circulating blood ecosystem. Phospholipids and lipid components of LDL inhibit infectivity and hemagglutination of rhabdoviruses, probably because of structural similarity between such compounds and the receptors for viruses in cell membranes.^190,191 In the case of vesicular stomatitis virus, phosphatidylinositol, phosphatidylserine and GM3 ganglioside show inhibitory activity.¹⁹² What are the mechanisms of action of such lipid moieties? Some light on this question is shed by studies of Mastromarino and colleagues¹⁹³ in which removal of negatively charged molecules from membrane lipids by enzyme treatment significantly reduces their inhibitory activity, suggesting that electrostatic interactions play important roles in viral cell membrane dynamics. It seems highly likely that similar electrostatic roles involving platelets, monocytes and other elements in circulating blood ecology will also be discovered in the future.

Granulocyte Clumping, Membrane Damage, and Lysis in AA Oxidopathy
The granulocyte is usually dismissed as inconsequential in discussions of atherogenesis. This surprises us for two reason: 1) we observe morphologic evidence of oxidative damage to granulocytes in AA oxidopathy with high frequency in patients with IHD; 2) it is known that granulocytes produce toxic oxidative species that degrade other intracellular and extracellular molecular species, inflict peroxidative injury to cytoplasmic and organelle membranes, enhance polymorphonuclear leukocyte-endothelial adhesion, and increase microvascular permeability.^194-200 Evidently, all of those factors can initiate, perpetuate and intensify oxidative phenomena that cause oxidative coagulopathy and AA oxidopathy and may result in IHD. Some oxidizing molecular species elaborated by granulocytes increase capillary permeability and enhance granulocyte-endothelial adhesiveness.²⁰² It seems odd to us that the cell known to play initial and critical roles in oxidative tissue injury is ignored in conditions characterized by oxidative injury to the circulating blood that results in atherogenesis. We recognized that granulocytes would be found to play a central role in atherogenesis when the molecular dynamics of this cell in atherogenesis are eventually investigated. This, indeed, is beginning to happen.
 
The granulocyte, like the erythrocyte, is a victim of the current infatuation of cholesterol enthusiasts with cholesterol. Our microscopic findings show that granulocytes play pivotal roles in initiating and perpetuating oxidative cascades in the circulating blood. In freshly prepared, unstained peripheral blood smears of healthy subjects, we observe granulocytes as hunter cells that move like crabs on the ocean floor, their locomotion provided by streaming of their granules into little protrusions of their cytoplasm. These cells continuously change their shapes as they explore their microenvironment. Not uncommonly, we visualize active phagocytosis of bacteria and cellular debris by such cells. In AA oxidopathy, the earliest change involving granulocytes is loss of locomotion—the cells lie limp in pools of plasma, with diminished or absent granular streaming. In later stages, granulocytes exhibit clumping. As in the case of erythrocytes, some granulocytes in more advanced cases of AA oxidopathy show blurring of membranes while others appear as ghost outlines of cells. Eventually, badly damaged granulocyte show disintegration of segments of their walls, degranulation and lysis.
 
The cytoplasmic granules of human granulocytes are rich in many enzymes including proteases, such as elastase, which are capable of degrading proteins in intracellular as well extracellular fluids.²⁰² Oxidative cell membrane injury may be expected to result in escape of proteases from granulocytes into the circulating blood. The destructive capacity of granulocytes represents an exaggerated physiologic response in which bursts of potent oxidative molecular species are produced during inflammatory and repair responses. Specifically, hydroxyl radical (OH.) derived from superoxide radical (O2-) produced by granulocytes are a major cause of cellular injury. Granulocytic myeloperoxidase generates hypochlorite radicals when exposed to H2O2 following phagocytic activation.²⁰³ Hypochlorite, in turn, oxidizes protease inhibitors, thus leading to increased proteolytic tissue damage.

 Granulocytes play a central role in the generation and function of oxidative species that control cellular signaling, regulate mediators of inflammatory and repair responses, and influence migration and replication of inflammatory cells.^204-207 A spate of recent gene-activation studies show evidence of the involvement of granulocytes in atherogenesis. Transcription of many atheroscleroses-related genes is augmented by oxidant-sensitive regulatory pathways involving nuclear factor kB (NF-kB).²⁰⁷ Specifically, exposure to superoxide radicals produced in granulocytes—and to lesser degrees in other cells—activates the NF-kB regulatory complex,^206,207 which, in turn, triggers transcription of genes that encode for a variety of proteins including leukocyte adhesion molecules, chemotactic cytokines and enzymes that regulate cellular and matrix metabolism.²⁰⁷ Indirect evidence of the relevance of granulocytic factors in coronary artery disease has been shown by Tanaka et al.²⁰⁴ who documented activation of vascular cells and leukocytes in the rabbit aorta after balloon injury. Direct evidence for activation of NF-kB in experimental injury has recently been shown by Lindner et al.²⁰⁹ Recent findings of Tardif et al.⁹⁰ that probucol reduces the incidence of restenosis after coronary angioplasty is consistent with such considerations, since the drug is a potent antioxidant and would be expected to protect coronary arteries traumatized by the angioplasty procedure from granulocytic oxidative bursts.