Platelet Aggregation and Lysis in AA
Oxidopathy
The role of platelets in atherogenesis and coronary thrombosis has drawn
much—and persistent—attention.210-222 Yet, the atherogenic role of
oxidatively damaged platelets in the circulating blood is ignored, just as the atherogenic
consequences of oxidatively damaged circulating erythrocytes and granulocytes are
neglected in deliberations of atherogenesis. In pathogenesis of atherosclerosis, the role
of platelets is usually limited to the circumstances under which platelets adhere to
endothelium or subendothelial stroma. This shifts the focus—to a great detriment to
clear understanding of the pathogenesis of IHD—from initial molecular oxidative
events taking place in the blood ecosystem to subsequent cellular oxidative events
occurring in the vessel wall ecosystem.
In freshly prepared, unstained peripheral blood smears examined with a
high-resolution, phase-contrast microscope, platelets appear as dark, round-to- ovoid, structureless bodies with poorly visualized granules, and without well-delineated plasma
membranes. There is little or no tendency toward clumping and the plasma in their vicinity
shows no evidence of congealing. Indeed even when smears are allowed to stand for 15 to 30
minutes, platelets remain discrete and do not cause congealing of fields of plasma that
surround them in the central portions of the smears. (The peripheral portions of such
smears often show early platelet clumping due to oxidative stress caused by exposure to
the ambient oxygen.) Familiarity with the range of platelet morphology observed in health
is essential before an observer can meaningfully interpret platelet changing seen in AA
oxidopathy and oxidative coagulopathy.
In subjects with known atherogenic risk factors—especially in
smokers, uncontrolled diabetics and those with chronic inflammatory conditions—we
observe evidence of variable damage to platelet membranes and degranulation. The platelets
in AA oxidopathy aggregate, change shape, degranulate and release various thrombogenic and
atherogenic factors. Not unexpectedly, most platelet aggregates and clumps are surrounded
zones of plasma congealing of variable widths. In more advanced stages of AA oxidopathy,
platelet membranes become indistinct and lysis occurs. Parenthetically, we might add that
we also observe similar damage in patients with disabling chronic fatigue,
fibromyalgia,
chemical sensitivity and a host of acute autoimmune disorders. Such changes are only
rarely seen in apparently healthy subjects.
One of us (MA) established the oxidative redox nature of platelet
aggregation and clot formation by addition of ascorbic acid and ethylenediaminetetraacetic
acid (EDTA) to platelet aggregates induced by oxidizing agents such as collagen,
epinephrine, ADP and ristocetin. We observed that both ascorbic acid and EDTA can readily
break up platelet aggregates formed by addition of various aggregating agents.13
Those observations support our view that platelet aggregation and clot formation are
oxidative phenomena and that antioxidants ascorbic acid and EDTA caused dispersal of
platelet aggregates by protecting the platelet membranes from the oxidant stress.
Interestingly, both ascorbic acid and EDTA failed to break up platelet aggregates caused
by collagen, indicating a stronger—and perhaps irreversible—effect of collagen
on platelet aggregation. From a teleologic perspective, it may be argued that collagen
exerts a stronger aggregating influence than epinephrine because circulating platelets are
exposed to collagen under more threatening conditions (bleeding from trauma to vessel
walls) rather than to epinephrine (a common hyperadrenergic state created by lifestyle
stressors).
Endothelial cells and platelets repel each other by their nonthrombogenic character—by their surface charges as well as their ability to
generate antithrombotic molecules such as heparin and prostacyclin.220 Thus,
adhesion of platelets to endothelial cells is prevented under ordinary conditions. Such
electromagnetic and molecular conditions, however, are threatened continuously by the
normal oxidative stress in healthy circulating blood. In states associated with
accelerated oxidative injury, the normal nonthrombogenic capacity of platelets and
endothelial cells is exceeded and platelets begin to agglutinate and adhere to endothelial
cells. Examples of conditions of accelerated oxidative stress include catecholamine surges
that accompany lifestyle stresses, hypercholesterolemia, denuding endothelial injury
caused by intra-arterial catheters, and anastomotic sites of bypass surgery. Under such
conditions, injury to platelets triggers chain reactions of oxidative coagulopathy, first
in the blood and subsequently in the vascular wall affecting all four lines of cells
involved in atherogenesis—endothelial cells, monocytes/macrophages, myocytes, and yet
more platelets.221-226 Platelet degranulation releases several growth factors,
including platelet-derived growth factor (PDGF),215-217 epidermal growth
factor,227 nitric oxide,228 and transforming growth factor-beta.228
Some of these growth factors are powerful mitogens. But generation of all such growth
factors is initiated by direct oxidative stress on platelets.
What is the common denominator in all platelet factors that are
associated with IHD? Evidently, it is accelerated oxidative injury to elements in the
circulating blood that leads to oxidative coagulopathy and AA oxidopathy. Again, as for
erythrocytes and granulocytes, the patterns of oxidative damage to the components of the
vascular wall that lead to plaque formation—and which have claimed enormous sums of
research funds without significant benefit to those who suffer from IHD—clearly are
consequences of changes in the circulating blood.
Diaphanous Congealing of Plasma
We observed diaphanous zones of plasma congealing surrounding platelets,
fragments of leukocytes, and fungal organisms. In many cases we observed areas of plasma
congealing without any involvement of platelets, leukocytes and fungal organisms. That
some free radical activity exists in plasma in health must be accepted on teleologic
grounds alone. Such oxidative stress is generated by normal metabolic activity of red and
white blood corpuscles as well as of platelets, oxidation of catecholamines, enzymatic
glucose breakdown, nonenzymatic autoxidation of blood glucose in hyperglycemic states, and
mechanical shearing stress on endothelial cells. Furthermore, zones of plasma congealing
and microclots produced by physiologic redox dynamics may be expected to be dissolved as
soon as they form by normal plasma fibrinolytic activity. Notwithstanding such physiologic
fibrinolytic activity, some free radical damage to the endothelium and subendothelial
matrix would be expected to ensue. Indeed, the presence of fatty-streak lesions in
children attests to the existence of such insidious and clinically silent oxidative
coagulopathy.
It is to be expected that normal oxidative stresses on blood plasma are
markedly increased during a host of pathologic states of the cardiovascular system, as
well as of other body organ ecosystems, accompanied by accelerated oxidative injury. This
includes advanced IHD, unstable angina, congestive heart failure, cardiac arrhythmias,
hypertensive crises, hyperglycemia, and during smoking.
Lumpy Coagulum and Fibrin Needles
Intravascular coagulation has long been assumed to be an uncommon and
potentially life-threatening state. Our high-resolution, phase-contrast microscopy
observations of peripheral blood in a host of cardiovascular and non-cardiovascular
entities challenge this assumption. In health, plasma in peripheral blood smears appears
as clear liquid that bathes cells. In states of accelerated oxidative molecular injury,
damaged plasma proteins begin to congeal, and such zones of clotted plasma spread as thin
diaphanous films. As the oxidative process advances, cross-linked fibrin appears as
filamentous and lumpy coagulum. Some platelets can usually be recognized trapped within
filamentous and lumpy fibrin deposits, undoubtedly contributing oxidized phospholipids and glycolipids to the protein coagulum. Such needles and masses of oxidized, coagulated
proteins and peroxidized lipids grow by triggering the chain reactions of plasma lipid
peroxidation and protein coagulation. We have consistently documented the presence of
fibrin needles and lumpy coagulum of protein in freshly prepared and unstained blood
smears in states of accelerated oxidative damage. By comparing peripheral blood morphology
before and after intravenous infusions of EDTA and ascorbic acid, both administered with
magnesium, we have repeatedly observed dissolution of fibrin needles and lumpy protein
after the infusions in cases in which such evidence of oxidative coagulopathy was clearly
discerned.
Microclots
The presence in circulating blood of microclots formed by oxidative
stress of normal blood ecology, and an excess of such clots in states of accelerated
oxidative stress, may be reasonably deduced from the foregoing discussion of redox
dynamics of plasma components and blood corpuscles in health and disease. Congealing of
plasma, erythrocyte and leukocyte membrane damage and platelet clumping may be expected to
add to the oxidizing capacity of blood by triggering fibrinogenic and lipid peroxidation
chain reactions. Furthermore, such changes may be expected to initiate oxidative chain
reactions, thus increasing oxidative stress and enlarging zones of plasma congealing into
microclots. We document such progressive changes of AA oxidopathy.
Microplaques
A natural consequence of oxidant microclots—oxidative coals, in our
terminology—circulating in blood would be for them to grow in size as the plasma at
their periphery continues to congeal and as an increasing number of platelets and other
blood corpuscles are entrapped into or stick to them. With ongoing oxidative stress, such
microclots coalesce to make yet larger and lumpier microclots. With time, such loosely
bound microclots are compacted in form layered structures with dead and dying cells and
other necrotic debris trapped between layers of fibrin that we call microplaques. Such
microclots and microplaques float in the bloodstream as simmering oxidative coals,
lighting up oxidative fires and inflicting further oxidative damage to blood corpuscles,
endothelial cells and subendothelial collagen matrix wherever the lining cells of the
vascular lumen have been denuded by the shearing mechanical stress of circulating blood.
We have observed microclots grow into microplaques that measure as much as several hundred
microns.
All oxidants in circulating blood trigger oxidative coagulative
phenomena involving blood corpuscles and plasma contents. Our clinical and high-resolution
microscopic observations lead us to consider the following groups of causes of accelerated
oxidative stress on the circulating blood that lead to oxidative coagulopathy:
1. Adrenergic hypervigilence associated with
lifestyle stressors
2. Rapid glucose-insulin and adrenergic shifts
3. Mycotoxicity and, to lesser degrees, toxins from other microbes
4. Increased oxidizability of blood associated with obesity
5. Diminished dietary intake of natural antioxidants
6. Increased body burden of prooxidants such as iron, copper and mercury
7. Inflammatory factors
8. Infectious agents
9. Excess of oxidized and denatured lipids
10. Autoimmune factors
11. Oxidative stress of cigarette smoking
12. Hyperhomocysteinemia
13. Mechanical shearing stress associated with hypertension
AA Oxidopathy and Fungemia
There are four important questions here:
1. How often are fungal organisms seen in the circulating blood of
nonfebrile ambulatory persons?
2. What roles do such organisms play in the pathogenesis of
oxidative coagulopathy and AA oxidopathy?
3. What are the possible mechanisms of action of mycotoxins and
other fungal proteins?
4. What roles do fungal organisms play in the inflammatory and
autoimmune processes that are known to be atherogenic and involved in other aspects of
IHD?
As to the first question of how frequently fungal
organisms may be observed in afebrile ambulatory patients, there is wide divergence of
opinion among those who routinely use high-resolution (15,000 x) phase-contrast microscopy
and those who never use such technology. We have documented the presence of fungal
organisms in peripheral blood of severely immunocompromised individuals with high
frequency (over 95%).229 As a part of our study of the phenomena of oxidative
coagulopathy and AA oxidopathy, we also examined the peripheral blood smears of 50
consecutive patients with advanced IHD (including those recovering from angioplasty and
coronary bypass operations) and detected the presence of fungal organisms in many
microscopic fields in 19. Identification of specific fungal species cannot be done with
such microscopy. However, employing anticandida antibodies labeled with horseradish
peroxidase, we have documented the presence of Candida species in peripheral smears in
some cases.230,231 We have previously published the specificity characteristics
of the anti-candida antibodies we employed in such studies.232,233 Such
observations may be challenged by those unfamiliar with high-resolution microscopy on the
ground that if true fungemia did exist in such patients, they would be critically ill.
This requires further comment.
The clinical distinction between benign bacteremia and potentially
life-threatening septicemia is well recognized; the former occurs after tooth brushing and
is clinically insignificant. It is noteworthy that no such clinical distinction is made in
the prevailing medical thinking between insidious and clinically silent fungemia and
potentially life-threatening fungal invasion of the bloodstream. Fungemia, the presence of
fungi in circulating blood, is always considered a serious pathologic entity. This is
clearly erroneous in view of the direct evidence to the contrary that we present here.
Regrettably, many physicians who have not taken the time to learn the use of
high-resolution microscopy—and hence are uninformed about the prevalence of fungal
organisms in the peripheral blood of immunocompromised individuals—make irresponsible
and derogatory statements about those who use such technology. Indeed, some licensing
boards controlled by such uninformed physicians have taken serious disciplinary actions,
including suspension of medical licenses, against holistic practitioners who diagnosed
fungemia with high-resolution phase-contrast microscopy and treated clinical
yeast
syndromes.234
Fungemia, Mycelia Formation and Fungal
Budding
In figures 27 through 30 (please e-mail
for availability of journal reprints), we illustrate the replication, mycelia
formation and fungal budding in peripheral blood smears observed over a period of five
hours for two reasons: 1) to provide additional proof that the bodies we recognize as
fungal organisms are indeed fungi (shown by their ability to form mycelia and the ability
of the mycelia to show budding); and 2) to document the rapidity with which fungal
organisms multiply as oxygen tension falls and acidity increases in their
microenvironment—the two conditions under which fungi would be expected to grow
luxuriantly.
As to the second question concerning the possible roles of fungal
proteins and mycotoxins in the pathogenesis of oxidative coagulopathy and AA oxidopathy,
we illustrate some of the observable phenomenon of zones of plasma congealing surrounding
fungal organisms.. We observed this phenomenon to occur within ten to sixty minutes in
almost all instances in which we studied the morphology of fungal organisms continuously
in freshly prepared unstained peripheral blood smears. We also observed fungal spores to
germinate within one to ten hours in most such cases. The zones of plasma congealing
surrounding fungal organisms increase in area, trap platelets and cellular debris, and
grow into microclots, and finally into micro-plaques. Such findings suggest that fungal
organisms play a role in the pathogenesis of oxidative coagulopathy and AA oxidopathy. We
return to this subject later in this article.
If fungemia occurs frequently in chronic immune disorders, why can't
the fungal organisms be cultured from blood in such cases? This is a valid question. We
have addressed this issue at length elsewhere.235 It is noteworthy that
negative blood cultures are frequently seen in patients with documented invasive tissue
fungal infections. In one study of such patients, a Candida enzyme called enolase was
detected in 42 percent of patients with proven tissue candidiasis.236
The third and fourth questions concern the possible molecular mechanisms by
which fungi cause AA oxidopathy and might play etiologic roles in the pathogenesis of IHD.
We return to this subject after discussing AA oxidopathy in relationship to the known
molecular dynamics of IHD.
AA OXIDOPATHY HYPOTHESIS IS CONSISTENT WITH
ALL KNOWN MOLECULAR DYNAMICS OF IHD
Molecular dynamics that preserve the
clotting-unclotting equilibrium (CUE) of life are marvels of biology. An elaborate system
of coagulative proteins, fibrinolytic enzymes and inhibitors of fibrinolysis exists in the
circulating blood that prevents clotting-unclotting disequilibrium (CUD) in health and
causes prompt clotting of blood when the integrity of the vascular wall is breached. Our
microscopic findings indicate that oxidative coagulopathy is the morphologic expression of
initial oxidative disequilibrium of redox in the circulating blood (early changes of CUD).
The broader range of changes of AA oxidopathy involving all circulating blood elements
(erythrocytes, granulocytes, lymphocytes, platelets, and plasma components), as well as
elements of the vascular wall, myocardial cell membrane, and conducting system are the
later events (full expression of CUD). We regard atherosclerosis as the structural tissue
response to chronic and insidious AA oxidopathy.
We have briefly reviewed the basic aspects of spontaneity of oxidation
in nature and molecular duality of oxygen and have presented a host of morphologic
patterns of oxidative coagulopathy and AA oxidopathy. Now we address the essential issue
of how consistent our proposed AA oxidopathy hypothesis is to all known molecular dynamics
of IHD. We follow that review with a discussion of the pathogenesis of cell and plasma
membranes permeability dysfunctions (leaky cell membrane dysfunction) which is an integral
part of AA oxidopathy. Finally, we present evidence for our view that dysregulations of
cholesterol and related lipids are the consequence and not the cause of pathophysiologic
derangements that result in AA oxidopathy and ischemic heart disease. The table on the
following page gives a listing of the pro-oxidant factors that contribute to pathogenesis
of AA oxidopathy and the antioxidant elements which normally arrest oxidopathy and have
been—or may be—clinically employed to reverse ischemic heart disease.
In Part II of this article, we will address the issue of how well
our hypothesis explains all known clinical risk factors of IHD.
