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thesis entitled

PERSISTENT BENEFICIAL EFFECTS OF A NOVEL NONANTI:
COAGULANT HEPARIN (Q4 1892) ON CARDIOVASCULAR
AND HEPATIC FUNCTIONS AFTER TRAUMA-"RRHAGE

AND RESUSCITATION

presented by
John Paul Repros

has been accepted towards fulfillment
of the requirements for

Master's degree in Suggry

32M

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Major prof7t/

Date January 15, 1996

 

0.7639 MS U is an Affirmative Action/Equal Opportunity Institution

 

 

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University

 

 

 

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MSU I. An Affirmative Action/Equal Oppommlty lnutltdlon
Wan-9.1

PERSISTENT BENEFICIAL EFFECTS OF A NOVEL NONANTICOAGULANT
HEPARIN (GM1892) ON CARDIOVASCULAR AND HEPATIC FUNCTIONS
AFTER TRAUMA-HEMORRHAGE AND RESUSCITATION

By
John Paul Kepros

A THESIS

Submitted to
Michigan State University
in partial firlfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Surgery
1 995

ABSTRACT

PERSISTENT BENEFICIAL EFFECTS OF A NOVEL NONANTICOAGULANT
HEPARIN (GM 1892) ON CARDIOVASCULAR AND HEPATIC FUNCTIONS
AFTER TRAUMA-HEMORRHAGE AND RESUSCITATION

By

John Paul Kepros

Although a nonanticoagulant heparin, GM 1892, improves cardiovascular and
hepatocellular functions immediately after hemorrhage and resuscitation, it is not known
whether this effect persists. To study this, rats underwent trauma and hemorrhage. The
rats were then resuscitated with crystalloid plus GM1892, conventional heparin, or
normal saline. At 24 hours afier resuscitation, cardiac index, stroke volume, and total
peripheral resistance were determined. SGOT, liver water content and hepatic
microvascular blood flow were also determined. TNF, IL-6 and PGE2 were measured to
investigate the mechanism of the action of GM1892.

The results indicate that cardiovascular fiinction remains depressed at 24 hours
after resuscitation. Treatment with GM1892 or conventional heparin, however,
significantly improves this function and reduces hepatic damage and edema. Furthermore,
the mechanism of the beneficial effects of GM] 892 appears to be downregulation of
proinflammatory cytokine (TNF, IL-6) production. Thus, GM1892, which, unlike
conventional heparin, has no significant anticoagulant activity, appears to be usefiil for
maintaining cardiovascular function and hepatic integrity following trauma-hemorrhage

and resuscitation.

ACKNOWLEDGMENTS

I would like to acknowledge the hard work and help Irshad Chaudry, Ping Wang,
Alfred Ayala, Rao Kareti, Mark Gonnan, and Jim Harkema. I would also like to express
my thanks and appreciation to Mary Morrison, Zheng F. Ba and the other members of the
laboratory team without whose help this would not have been possible to complete.
Finally, I would also like to express my thanks to Michele Hurrell for her patience
throughout the writing of this thesis.

iii

TABLE OF CONTENTS

1. List of Tables ............................................................................................... v
2. List of F igum .............................................................................................. vi
3. Introduction ................................................................................................. l
4. Hemorrhagic Shock ..................................................................................... 2
5. Mediators .................................................................................................... 8
6. Pharmacologic Therapy in Shock ................................................................ 10
7. Heparin and GM1892 ................................................................................. 12
8. Hypothesis .................................................................................................. l6
9. Specific Aims .............................................................................................. 16
10. Materials and Methods .............................................................................. 17
11. Results ...................................................................................................... 28
12. Discussion ................................................................................................. 48
13. Conclusion ................................................................................................ 56
13. References ................................................................................................. 57

iv

LIST OF TABLES

1. Tissue water content ....................................................................................... 3 8

2. Microvascular blood flow ............................................................................... 40

LIST OF FIGURES

1. Chemical structures of heparin and GM1892 ......................................................... 15
2. In-vivo hemoreflectometer catheter placement ........................................................ 20
3. Schematic diagram of cardiac output measurement ................................................. 21
4. Cardiac output curve .............................................................................................. 22
5. Calculation of Vm and Km from the double reciprocal plot of l/dose vs. 1N0 ........ 25
6. Heart rate ............................................................................................................... 29
7. Mean arterial pressure ............................................................................................. 30
8. Cardiac index .......................................................................................................... 31
9. Stroke volume ........................................................................................................ 32
10. Total peripheral resistance ..................................................................................... 33
1 1. V0 ....................................................................................................................... 3 5
12. Glutarnic oxalacetic transaminase (GOT) ............................................................... 36
13. IL-6 ...................................................................................................................... 42
14. TNF ...................................................................................................................... 43
15. TNF as measured by enzyme linked immunoabsorbant assay (ELISA) ................... 44
16. PGE2 ................................................................................................................... 45
17. Mortality .............................................................................................................. 47
18. Summary of the mechanism of action of GM 1892 ................................................. 52

INTRODUCTION

Historically, steady improvement has been made in the treatment of trauma
patients. Primarily this has been in clarifying the problem (Simeone, 1963) and specifically
in areas of fluid resuscitation (Shires, 1961), systematic management (Committee on
Trauma, 1989), and nutrition (Dudrick, 1968). More recently, increased understanding of
soluble mediators and improved animal models of hemorrhagic shock have led toward
better understanding of shock at a cellular level including its effect of the immune system
(Chaudry, 1993a). Along with this, corresponding areas of potential pharmacologic
intervention have emerged (Harkema, 1992). This thesis will discuss the history of shock
including the treatment and recent investigation of soluble mediators up to the present
time. This will be followed by a study of the effects of a potential new therapeutic agent

for the treatment of trauma and hemorrhagic shock.

HEMORRHAGIC SHOCK
History

Shock was first recognized in the early 1700's and the term shock was first used in
1743. Shock has also been referred to as a ”rude unhinging of the machinery of life", and
"a momentary pause in the act of death". Most of the observations on injured patients
have been during wars and on the battlefield. Curiously, many illnesses including some
war wounds were treated by phlebotomy. It was in 1872 that Samuel D. Gross first
postulated that shock was a reaction to major injury (Sabiston, 1991).

Laboratory investigation of shock in the late 1800's showed that the likelihood of
shock increased with the severity of the injury and prior hemorrhage impaired the ability of
an animal to survive an injury (Sabiston, 1991). Experimental animals were found to have
a decreased central venous pressure and administration of intravenous saline improved
survival. The importance of the reduction of tissue damage first became evident during
World War I, when splinting of femur fi'actures reduced the mortality from 70% to 10%
for this injury. After World War I it was hypothesized that the systemic effects of shock
were caused by toxic products from tissue damage. Further research showed that one of
the toxic factors, the hydrogen ion was found to be responsible for metabolic acidemia and
cardiovascular failure. This was countered by an experiment which showed internal
hemorrhage would account for intravascular volume loss (Blalock, 1930); (Blalock,
1934)

During World War II, a dog model was used by Wiggers in the investigation of
hemorrhagic shock and led to the development of the concept of irreversible shock
(Wiggers, 1945). It was found that after a period of constant hypotension, the animal
would decompensate and reinfusion of shed blood would not be sufficient to resuscitate
the animal. Shires found that infusing large volumes of intravenous fluid would prevent

renal failure and that this was superior to plasma or blood alone (Shires, 1964). It was

2

3

then discovered that an interstitital fluid deficit was responsible for the large amounts of
fluid required for resuscitation. wring this time period, a large amount of research was
also performed by Moore on the effects of hemorrhage (Moore, 1965). On a cellular
level, it was discovered that the cell membrane function changes resulting in movement of
sodium and water into the cell. Attempts were made to localize the energy pathway in
shock and the site of breakdown (Schumer, 1968). This method of aggressive fluid
resuscitation resulted in many patients surviving an initial severe injury during the Vietnam
conflict. The patients then developed a respiratory distress syndrome secondary to an
interstitial edema. The invention of the pulmonary artery catheter revealed that this
interstitial edema was secondary to an increased vascular permeability and not by high
pulmonary artery pressures. Experiments showed that excluding one lung of an animal
from circulation prevented damage and supported the theory of toxic factor involvement
in the efl‘ects of hemorrhagic shock.

More recently, shock has been related to alterations in immune function which can
result in sepsis (Abraham, 1991). It has also been hypothesized that the irreversibility seen
in hemorrhagic shock may be caused by sepsis (Rush, 1989). Inflammatory cytokines are
increased in shock (Abraham, 1988) and contribute to the immune depression.
Translocation of bacteria from the intestine has also been thought to play a role in sepsis
resulting from hemorrhagic shock (Alexander, 1990). A hypothesis for the mechanism of
increased susceptibility to sepsis has been formulated using current information on shock
and mediators (Ayala, 1994). With better ventilatory support, more patients would
survive ARDS and were found to have failure of multiple organ systems (Baue, 1975).
Multisystem organ failure has become the most feared result of circulating mediators in
hemorrhagic shock (Anderson, 1990). Some evidence has been found pointing toward the
intestine as the source of cytokines in hemorrhagic shock (Deitch, 1994). The gut has
even been considered a central organ in the pathogenesis of hemorrhagic shock (\Vrlmore,

1988). The characteristics of different shock states are very similar and a common

4

pathophysiologic mechanism of generalized endothelial cell activation has been termed
systemic inflammatory response syndrome (Bone, 1992).

Different animal models have been used to study the contribution of toxic factors
to the effects of hemorrhagic shock (Bacalzo, 1971). Initially, fixed volume models were
used in which a fixed volume of blood was removed from the animal to produce
hemorrhagic shock. A major drawbacks of this model is that the amount of blood
removed depends on the hydration state of the animal. Attempts to develop a fixed volume
animal model with an untreated mortality of approximately 50% have been difficult
(Collins, 1969). Anesthesia also causes problems in animals models because most
anesthetic agents depress cardiac function to some degree and also decrease the metabolic
demands of the body thereby altering the response to hemorrhage. Because of the
beneficial effect of heparin on animals undergoing hemorrhage, the ideal model would also
be nonheparinized. Cardiac output has been measured with radiolabeled microspheres,
thermodilution, electromagnetic flowmetry, and F ick oxygen method. Microspheres
require blood sampling and provide only a limited, interrupted measurement of cardiac
output. Thermodilution is difiicult to perform accurately in small animals such as the rat
because of the difficulty in controlling the temperature of the injectate. Electromagnetic
flowmetry requires thoracotomy. Indocyanine green measurement with a fiberoptic
catheter allow repeated accurate measurements of cardiac output without blood sampling
and also allows measurements of hepatocellular firnction. For experimental purposes
trauma can be broken down into two separate events, the initial tissue trauma, and
hemorrhage. Many animal models have been described throughout the history of the
study of hemorrhagic shock (Chaudry, 1993b). Because heparin maintains microvascular
patency during and alter hemorrhage, it is recommended that a clinically relevant model
not be heparinized (Rana, 1992). Taking into account reproducibility, predictability, and
expense, one animal model in particular, a non-heparinized, non-anesthetized, fixed

pressure rat model has been developed and used extensively (Chaudry, 1989). With this

5

model it has been possible to assess changes in cardiac output, hepatocellular function, and
circulating blood volume successfully as sensitive indicators of the pathological effects on
organs and homeostasis during as well as following the onset of hemorrhage (Wang,
1991a). A nonheparinized model is used in the present study because of the reported
beneficial effects of heparin on animals which have undergone hemorrhagic shock (Rana,
1992).

Events of Hemorrhagic Shock

Hemorrhagic shock results fi'om a loss of circulating blood volume which leads to
decreased perfusion of the nonvital tissues and vital organs. Anaerobic metabolism soon
follows from decreased perfirsion of the large nonvital tissue cell mass. Hormonal
mediators such as catecholamines are secreted and baroreceptors are stimulated producing
increased peripheral resistance.

The cellular response to hypoperfusion results in anoxia and cellular starvation.
Subsequently, anoxia limits the amount of substrate available to the cell. Severe and
prolonged hemorrhage produces a cascade of events which can ultimately lead to
increased susceptibility to sepsis, multiorgan failure and death. The mechanism for the
immune suppression is felt to be multifactorial and includes early systemic mediators,
decreased ATP levels, Ca++ alterations, and inflammatory mediator release (Chaudry,
1993a). The immediate hemorrhage is closely followed by regional hypoxia and alteration

in cellular firnctions. Directly or indirectly this results in the increased release of tumor

necrosis factor (TNF) from macrophages and this is followed by increased PGEZ

synthesis. PGE2 depresses macrophage and lymphocyte functions finally resulting in

increased susceptibilty to sepsis (Chaudry, 1993 a). Interleukin-6 is also elevated during
shock and may be usefirl as a marker of severity of shock (Damas, 1992).
Tumor necrosis factor in large doses has been found to reproduce the clinical

syndrome observed in shock (Tracey, 1986) as well as being an important mediator of the

6

inflammatory response cascade (Harkema, 1992). Prostaglandin E2 has been shown to

have important properties of immunosuppression and inflammation (Stephan, 1988).
Hemorrhage has been shown to decrease the ability of macrophages to present antigen
(Ayala, 1992).

There is evidence for prolonged depression in microvascular blood flow following
hemorrhage and resuscitation (Wang, 1989) as well as cardiac fimction (Wang, 1991a),
and hepatocellular function (Wang, 1990a) which persist despite fluid resuscitation.

Most mechanisms for the complication of multiple system organ failure from
hemorrhagic shock include cytokine release from macrophages (Border, 1988). The
mechanism of defective antigen presentation has also been studied (Ertel, 1991).
Translocation of intestinal bacteria continues to be examined as one of the most likely
origins of infectious agents in sepsis seen alter trauma (Border, 1987). In animal models,
post-shock cultures were normal rat enteric flora (Koziol, 1988). In fact, some have
considered the gastrointestinal tract to be the "motor" of multiple system organ failure.
(Meakins, 1986). Susceptibility to infection has also been found to persist despite fluid
resuscitation and antibiotics (Livingston, 1987). Despite this work, there are still many
questions regarding the precise events that relate hemorrhage to multiple organ failure
(Carrico, 1993).

Fluid Intervention in Hemorrhagic Shock

Fluid resuscitation is the primary therapy treatment for hemorrhagic shock. During
acute hemorrhagic shock, crystalloid infusion restores cardiac preload and helps to prevent
firrther ischemic damage and acute renal failure. Crystalloid resuscitation alone, however,
does not restore or maintain immune function (Ayala, 1992), cardiac output,
hepatocellular fiinction, microvascular blood flow, or glomerular filtration rate following
such conditions (Wang, 1991a). Small-volume, slow-rate saline infusion may produce

physiologic benefits that are independent of arterial pressure (Lilly, 1992).

7

Colloid solutions are useful in prehospital treatment of patients with hemorrhagic
shock where it is not possible to give large amounts of intravenous fluids. Colloid
administration provides a greater intravascular volume expansion than crystalloid for the
same volume but this efl‘ect is transient as shock is associated with an increased
permeability of the vascular endothelium and their is leakage of the colloid into the
interstitial space (V elanovich, 1989). Colloid solutions combined with hypertonic saline is
also much more effective than crystalloid solutions (Holcrott, 1984).

Administration of blood for the treatment of hemorrhagic shock is controversial.
Healthy animals have been shown to be able to tolerate a hematocrit as low as 15% as
long as there is adequate intravascular volume (Wilkerson, 1988). The decision to give
blood is usually determined by the patency status of the patient's coronary arteries. The
heart requires approximately 50% of the oxygen delivered to it and is the first organ to

become ischemic with a falling hematocrit.

 

MEDIATORS

Hemorrhage is responsible for the release of chemical mediators into the
bloodstream that affect the response to shock. A variety of mediators are released
including complement, hormonal mediators, cytokines (F ong, 1990), and eicosanoids.

Both the classic and alternative pathways can activate the complement cascade.
This activation generates anaphylatoxins which affect the hemodynamic system. C3a
stimulates histamine release, increases capillary permeability and causes smooth muscle
contraction. C 5a also increases capillary permeability, stimulates degranulation of mast
cells and is also a chemotactic factor (F eldbush, 1984).

Hormonal mediators released in response to hemorrhage are primarily released
acutely and are directed at compensating for the loss of intravascular volume. Some of
the hormones released include cortisol, anti-diuretic hormone (ADH), epinephrine,
norepinephrine, and aldosterone. Endorphines are also released in response to
hemorrhage from the pituitary gland (Moore, 1965). Trauma stimulates increased
secretion of CRF, ACTH, and cortisol. The result is increased cortisol levels. The efi‘ects
of cortisol are on many organs. Cortisol appears to maintain euglycemia during stress by
its action on the liver. Cortisol does inhibit immunologic and inflammatory responses. It
inhibits the function of lymphocytes, monocytes, and polyrnorphonuclear cells.
Antidiuretic hormone has osmoregulatory, vasoactive, and metabolic effects.
Catecholamine secretion results in increased secretion of glucagon and deccreased
secretion of insulin. The hemodynamic effects of catecholamines include vessel
constriction, an increase in heart rate and myocardial contractility. Catecholarnines may

also be usefirl is assessment of overall severity of an injury (Davies, 1984).

9

Cytokines are a class of proteins which include the interleukins, tumor necrosis
factors, transforming growth factors and colony stimulating factors. Cytokines are
biologically active at very low concentrations and are synthesized by many different types
of cells in amounts that are dependent on severity and location of the stimulus. Beneficial
effects of cytokines include antimicrobial functions, wound healing, and activation of
immune responses (Sheppard, 1989). Ifcytokines are present systemically in high
concentrations, however, they can produce hypotension, tissue damage and a state that is
very similar to shock (Nuytinck, 1988). Tumor necrosis factor is produced by activated
macrophages and causes neutrophil activation and increased vascular permeability. It also
causes hypotension, lactic acidosis and pulmonary failure (Tracey, 1986). TNF also is
involved in the production and release of leukotrienes and prostaglandin E2 (Aggarwal,
1985). TNF induces ARDS when infused into animals (Ferrari-Baliviera, 1989). TNF has
also been shown to produce shock and tissue injury when given alone (Tracey, 1986).

Free radical injury also is involved in shock. After an episode of ischemia, elevated
hypoxanthine and xanthine levels may develop and form the superoxide radical which can
lead to cell and endothelial damage. Other reported effects of free radical injury include
microvascular permeability, granulocyte adhesion and arteriolar vasoconstriction. (Parks,
1983.)

Eicosanoids have many diverse effects on the physiologic and immune systems
(Fletcher, 1979). Prostaglandins and leukotrienes are the primary metabolites of
arachidonic acid that are involved in shock. These metabolites are released fi'om
macrophages in response to hemorrhage (Bankhurst, 1981). Prostaglandin levels are also
elevated in response to endotoxin shock (Anderson, 1982). Leukotrienes have also been
implicated as mediators in tissue trauma (Denzlinger, 1985). Thromboxane is stimulated
by regional ischemia which occurs in hemorrhagic shock (Lelcuk, 1984). Prostaglandin

F? also deprsses antigen presenting cell function of peritoneal macrophages (Stephan,

1988)

 

PHARMACOLOGIC THERAPY IN SHOCK

Different therapeutic agents have potential for use in hemorrhagic shock. These
agents are directed at the various factors which appear to contribute to the pathologic
processes involved in shock. With optimization of systematic management and fluid
resuscitation, recent efforts have been on intervening at the cellular level with
pharmacologic agents (Harkema, 1992). Certain pharmacologic agents such as
cyclooxygenase inhibitors, platelet activating factor antagonists, calcium antagonists,
cytokine antibodies or antagonists, may be needed during and following resuscitation to
restore the immunological functions, as well as improve organ function (Harkema, 1992).
Preheparanization of animals produces various beneficial effects, such as improvement of
cell and organ firnction as well as survival time of experimental animals following adverse
circulatory conditions (Rana, 1992). Administration of conventional heparin following
trauma-hemorrhage and resuscitation, significantly improves organ function and
microcirculation (Wang, 1994a). ATP-MgClz has been used to correct the depression of
adenosine triphosphate (ATP) production (Chaudry, 1981). Pentoxifylline may be
efl‘ective in improving microcirculation or downregulating neutrophil response (Coccia,
1989). Pentoxifylline may function by suppressing TNF gene transcription which gives
some insight and direction for the development of other chemical therapeutic agents
(Doherty, 1991). Calcium channel antagonists may be effective in restoring calcium
homeostasis within the cell. In animal models, calcium channel blockade has been shown
to improve myocardial and cellular function (Hess, 1983). Diltiazem may exert its effect
by resoring cytokine and interferon-y synthesis (Meldrum, 1991). Monoclonal antibodies
to inflammatory cytokines or their receptors appear to have some beneficial effects. A
receptor antagonist to IL-1 has been found to improve survival after endotoxemia in
animals (Alexander, 1991). Cytokine administration may also be of benefit in

desensitization to the negative influence of cytokines. Because of the critical role of

10

ll

cytokines in hemorrhagic shock different applications have emerged (Dinarello, 1993). As
mentioned, TNF has been found to reproduce the clinical effects seen in shock. It is often
thought of as the primary mediator in shock and thus blocking of TNF has been a primary
goal of many investigators because of the potential clinical value. Specific blockade of
TNF with neutralizing antibodies or soluble receptors reduces mortality and severity of
disease. The results are similar for blockade of IL-1 (Dinarello, 1993). TNF induced
mortality is also reversed with administration of cyclooxygenase inhibitors (Fletcher,
1993). Administration of anti-TNF antibodies has been found to prevent shock in animals

that are bacteremic (Tracey, 1987). Interferon-y may be helpful in reducing the spread of

 

infection and decreasing the susceptibility to sepsis (Harkema, 1992).

Administration of eicosanoids or their inhibitors may be beneficial by improving
systemic or regional blood flow as well as a number of other possible mechanisms
(Almqvist, 1982). Administration of prostacyclin and thromboxane A2 has been found to
moderate postischemic renal failure (Lelcuk, 1985). Dietary administration of n-3 fatty
acids has been shown clinically to reduce wound infections, shorten hospital stay , and
reduce mortality. Free radical scavengers may be effective by reducing the amount of
oxygen free radicals which have been implicated in the damage caused by ischemic injury.
Coenzyme Q10 may protect against cellular damage by preserving energy content,
preventing oxygen-radical injury and stabilizing membrane structure and function.
Agents which may protect against dysfunction in the peripheral vascular regulation
(adrenergic agents, angiogensin-converting enzyme inhibitors) have also been shown to
have beneficial effects. Naloxone, which is an opiate antagonist, reverses some of the
detrimental effects produced by endogenous endorphin release (Curtis, 1980). One of the
proposed pharmacologic agents that may be usefirl in hemorrhagic shock is a chemically
modified heparin (GM1892). Heparin and chemically modified heparin will be discussed
below.

HEPARIN AND CHEMICALLY MODIFIED HEPARIN (GM1892)
Heparin

Heparin had initially been used as an agent in small animal models to maintain the
patency of small venous and arterial cannulas used in hemorrhage models. Administration
of heparin prior to hemorrhage has been shown to improve cardiac output, hepatocellular
function and renal function. (Wang, 1990b). Heparin was found to have beneficial efl‘ects
on animal survival after hemorrhage but could not be used in clinical situations because its
anticoagulant effect precludes its use in trauma patients (Rana, 1992). Stembergh et. al.
demonstrated, however, that the anticoagulant effect and the beneficial effects in animals
were independent (Stembergh, 1993). Preheparanization of animals prior to hemorrhagic
shock has also been found to protect the microvasculature (Rana, 1992). Heparin has been
shown to improve survival in animal models of gram-negative septic shock (Griffin, 1990).

Heparin is released from baSOphilic granules of mast cells and leukocytes during
inflammatory reactions (Dziarski, 1992). During these conditions it has been found to
have several immunomodulatory effects. Lymphocytes have been found to have binding
sites for heparin (Parish, 1985). At clinically utilized doses it enhances the proliferative
responses of murine T cells in mixed lymphocyte reaction. It also enhances the generation
of cytotoxic T lymphocytes against allogeneic cells and histocompatible tumors (Dziarski,
1989). It also plays a role in the modulation of immunologic function (Lider, 1989).

Several of the other nonanticoagulant properties include modulation of smooth
muscle cell proliferation (Clowes, 1977), angiogenesis (F olkman, 1989), platelet function
(Gregorius, 1976), and acute inflammation (Carr, 1979). There is evidence to show that
heparin reduces vascular permeability (Hobson, 1988). It also has membrane stabilizing
effects (Srinivasan, 1968) as well as increasing superoxide dismutase production which
may decrease the toxic effects of free radicals (Karrison, 1988). Heparin may also

increase cGMP levels by increasing endothelial EDRF release (Stembergh, 1993).

12

13

The exact mechanism of the beneficial effects of heparin, however, is unknown.
Heparin and modifed forms of heparin have been shown to inhibit complement activation
in-vivo (W eiler, 1992). It has been hypothesized that the strong negative charge of
heparin and the increased release of tumor necrosis factor-binding protein by heparin
could be responsible for their beneficial effects (Lantz, 1991). The mechanism may be
related to the firnction of the endothelium which is preserved in preheparinized animals
(Wang, 1993). The highly negatively charged heparin molecule may also attach to red
blood cells and keep them dispersed in low blood flow situations (Chapra, 1967).

The elimination curve of conventional heparin is biphasic consisting of a rapidly
declining alpha phase with a half life of 10 minutes and after the age of 40, a slower beta
phase, indicating uptake in organs. The is no relationship between anticoagulant half-life
and concentration half-life indicating that there might be some protein binding of heparin

(Physicians Desk Reference, 1995).

GM1892

There are many different forms of nonanticoagulant heparin that have been
developed including low molecular weight heparins and other modified forms. GM1892 is
a nonanticoagulant heparin that was developed by Glycomed (Alameda, CA). GM1892 is
composed of the same natural saccharide residues found in heparin and is prepared from
the parent low molecular weight heparin (LMW) by selective O-desulfation using alkaline
conditions. This modification results in the loss of specific sulfate groups important to
antithrombin III (AT 111) binding and reduces the overall charge density of the molecule.
It has a molecular weight of 4-5 KDa and approximately 2% of the anticoagulant activity
of conventional heparin as measured by the activated partial thromboplastin time (Figure
1). It has approximately 60% of the antiplatelet activity of conventional heparin and no
growth factor activity. The ability of splenocytes to release IL-2 and IL-3 in response to

mitogen is markedly improved in hemorrhaged animals treated with GM1892 or

 

13

The exact mechanism of the beneficial effects of heparin, however, is unknown.
Heparin and modifed forms of heparin have been shown to inhibit complement activation
in-vivo (W eiler, 1992). It has been hypothesized that the strong negative charge of
heparin and the increased release of tumor necrosis factor-binding protein by heparin
could be responsible for their beneficial effects (Lantz, 1991). The mechanism may be
related to the function of the endothelium which is preserved in preheparinized animals
(Wang, 1993). The highly negatively charged heparin molecule may also attach to red
blood cells and keep them dispersed in low blood flow situations (Chopra, 1967).

The elimination curve of conventional heparin is biphasic consisting of a rapidly
declining alpha phase with a half life of 10 minutes and after the age of 40, a slower beta
phase, indicating uptake in organs. The is no relationship between anticoagulant half-life
and concentration half—life indicating that there might be some protein binding of heparin

(Physicians Desk Reference, 1995).

GM1892

There are many different forms of nonanticoagulant heparin that have been
developed including low molecular weight heparins and other modified forms. GM1892 is
a nonanticoagulant heparin that was developed by Glycomed (Alameda, CA). GM1892 is
composed of the same natural saccharide residues found in heparin and is prepared fiom
the parent low molecular weight heparin (LMW) by selective O-desulfation using alkaline
conditions. This modification results in the loss of specific sulfate groups important to
antithrombin III (AT III) binding and reduces the overall charge density of the molecule.
It has a molecular weight of 4-5 KDa and approximately 2% of the anticoagulant activity
of conventional heparin as measured by the activated partial thromboplastin time (Figure
1). It has approximately 60% of the antiplatelet activity of conventional heparin and no
growth factor activity. The ability of splenocytes to release IL-2 and IL-3 in response to

mitogen is markedly improved in hemorrhaged animals treated with GM1892 or

 

l4

conventional heparin compared to saline-treated animals. The capacity of splenic and
peritoneal macrophages to release IL-6 is also restored in the hemorrhaged animals that

receive GM1892 or conventional heparin (Zellweger, 1995).

Although GM1892 has been shown to improve hepatocellular filnction, cardiac
output, and microcirculation acutely (up to 4 hours) after trauma-hemorrhage and
resuscitation in rats (Wang, 1994a), it is not known whether this agent produces

persistent beneficial effects on organ function.

15

0803- 0803-

o
‘ 0503- > OH
1

NHSOS- NH SOS-
Heparin GM1892

Figure 1. Chemical structures of heparin and GM1892. GM1892 is
prepared by selective O-desulfation using alkaline conditions.

HYPOTHESIS
Administration of GM1892 improves cell and organ function in a chronic
trauma/hemorrhage model by improving microcirculation and by downregulating

proinflammatory cytokine (TNF, IL-6) synthesis Irelease.

SPECIFIC AINIS
1. To determine GM1892 improves cardiovascular and hepatocellular function in
a chronic trauma/hemorrhage model.
2. To determine the mechanism of action of GM1892.
3. To determine if administration of GM1892 improves survival in animals

subjected to trauma hemorrhage and resuscitation.

l6

MATERIALS AND METHODS
Experimental design

Eight animals were used for each sham group, saline group (control group),
chemically modified heparin (GM 1892) group, and heparin group. The animals in the
saline, GM1892, and heparin groups underwent trauma- hemorrhage and resuscitation as
described below. Twenty four hours after trauma-hemorrhage and resuscitation the
animals were anesthetized and heart rate, blood pressure were measured. Cardiac output
and hepatocellular firnction were measured using indocyanine green clearance. Blood
samples were obtained for TNF, interleukin-6 (IL-6), PGEZ , and aspartate
arninotransferase (AST) also known as serum glutamic-oxaloacetic transaminase (SGOT).
Major organs including the lungs, liver, kidneys, intestines, spleen and heart were removed
and weighed prior to and after dessication to quantitate tissue edema (Wang, 1992).

The mechanism of action of GM1892 was investigated by examining the possible
sites of action. Laser Doppler flowmetry was used to assess differences in surface
microcirculation of liver, kidneys, small intestine, and spleen between treated animals and
controls (Wang, 1990c). A bioassay for TNF described below was used to quantitate
TNF activity. The results of this were compared with the results of an ELISA quantitating
the total amount of antigenic TNF which detects bioactive and non-bioactive TNF thus
allowing an estimation of the presence of an inhibitor.

Animal model of trauma and hemorrhage

A nonheparinized model of trauma-hemorrhage and resuscitation in the rat was
used and which has been described previously (Chaudry, 1989). Male Sprague—Dawley
rats weighing 275-325 gm, were fasted overnight before the experiment but were allowed
water ad libitum. After methoxyflurane anesthesia, the animals underwent a 5 cm ventral
midline laparotomy to induce tissue trauma before hemorrhage. The abdominal incision

was then closed in 2 layers. Both femoral arteries were cannulated with polyethylene

l7

 

18

(PE)—50 tubing for bleeding or measurement of mean arterial pressure (MAP) and heart
rate (HR). MAP and HR were continuously monitored with a femoral catheter connected
to a manometer. The rats were bled to a mean arterial pressure (MAP) of 40 mmHg and
maintained at that pressure until 40% of the shed blood volume was returned as Ringer's
lactate (RL). This is the point or irreversibility in Wigger‘s model. The rats were then
resuscitated with 4X the volume of the maximal bleedout with RL over 60 minutes via the
femoral venous catheter. The animals received a single dose of GM1892 (7 mg/Kg),
heparin (7 mg/Kg), or an equivalent amount of normal saline intravenously in a bolus dose
at this time. Maximal bleedout was determined as the volume of blood withdrawn
following which the animal cannot maintain the MAP of 40 mm Hg unless some fluid was
returned to the intravascular space. Prehemorrhage and posthemorrhage hematocrit were
also measured. The shed blood was not returned to the animal. Control animals received
the same operation but were administered normal saline only instead of a pharmacologic
agent. Sham operated animals underwent the same surgical procedure but were not
hemorrhaged. All experiments were performed in adherence to the National Institutes of
Health Guide for the Care and Use of Laboratory Animals. This experiment was approved
by the Michigan State University committee on the care and use of animals.
Heart rate

Heart rate was determined by counting the number of pulsations of the
blood/saline interface in the cannulation tubing connecting the femoral artery to the
manometer. The number of pulsations was counted for a 15 second interval and the result

multiplied by 4 to give the number of beats per minute.

19

CO measurement
Cardiac output was determined by indocyanine green (ICG) dilution. A fiberoptic
catheter was placed in the left carotid artery to the level of the thoracic aorta (Figure 2).
A polyethylene catheter was placed in the right internal jugular vein. A carefully measured
quantity of ICG dye was injected into the internal jugular vein and the ICG concentrations
were measured continuously with the fiberoptic catheter and recorded digitally on a

computer disk through a data aquisition program (Figure 3).

The dose used for the calculation of cardiac output was the same dose as that used for the
measurement of hepatocellular function. The smallest dose (0.05 mg) gave the best results
for cardiac output as larger doses would result in higher blood concentrations near the
saturation level of the in-vivo hemoreflectometer. Dimensional analysis confirms that the
dose (mg) divided by the area under the curve (mg/L/min) gives the correct units for
cardiac output (L/min)(Figure 4). The cardiac output was then converted into a cardiac
index by dividing by the body weight of the rat.

The usual method or measurement is to place the fiberoptic catheter from the in-
vivo hemoreflectometer in the right sided carotid artery leaving it rest in the aorta.
Because of the small amount of distance between the junction of the carotid artery and the
inominate artery and the aortic valve, however, small movements in the rat can cause
movement of the fiberoptic catheter resulting in inaccurate measurements of ICG
concentrations. Because of this problem, it was decided to place the catheter in the left
sided carotid artery with the tip in the descending aorta. Using this method, there is
approximately 4 cm or more of room for the tip of the catheter. This creates a larger

distance for the tip of the catheter to be effective.

 

20

   

Figure 2. In-vivo hemoreflectometer catheter placement. Twenty four hours
following hemorrhage and resuscitation, a fiberoptic probe from an in-vivo
hemoreflectometer was placed in the left carotid artery approximately 4 cm to

place the tip in the descending aorta.

21

\l/ ICG dose

 

 

 

 

Fiberoptic catheter
(ICG concentration)
A
-—-) O ( Heart 1 - Q ___)

Figure 3. Schematic diagram of cardiac output measurement. An ICG dose
is given in the venous circulation close to the heart. The ICG concentration is
then monitored with a fiberoptic catheter in the aorta. The cardiac output is
determined from the area under the cardiac output curve.

 

22

CO = dose/area under curve

Figure 4. Cardiac output curve using a small dose of indocyanine green
(0.05mg).

 

 

 

time

23

Determination of hepatocellular function

Indocyanine green clearance was described in 1968 as being a nontoxic accurate
method of determining hepatocellular firnction (Bradley, 1968). It has since been shown
to be an accurate indicator of hepatic blood flow (Burczynski, 1987). Another advantage
of indocyanine green clearance is that it can also be used to determine circulating blood
volume concurrently (Busse, 1990). Indocyanine green is metabolized exclusively by the
liver and its metabolism reflects the hepatic blood flow and hepatocellular function
(Cherrick, 1960).

After a 24 hour recovery period for the animals, they were anesthetized with
pentobarbital and the right jugular vein was cannulated with PE-SO tubing. The left
carotid artery was isolated and a fiberoptic catheter was passed down the carotid artery to
the level of the aortic arch for the continuous measurement of ICG concentration. Three
doses of ICG (0.05, 0.10, 0.25 mg per rat) were administered with the smallest dose first.
After administration of the ICG, the concentration was recorded with an in-vivo
hemoreflectometer. For each dose, the nonlinear regression equation was calculated for
the relationship between ICG concentration and time according to the polynomial equation

[ICG] = exp(a + bt + ctz)
where [ICG] is the ICG concentration in milligrams per liter, t is the time in minutes, a, b,
and c are coefficients unique to each measurement. (Hauptman, 1991). V0 which is the
initial rate of change of ICG concentration was calculated by
V0 = b exp(a)
An explanation of the derivation of this is given in the discussion section below. The
initial velocity of clearance was converted from milligrams per liter per minute to

milligrams per kilogram per minute by
V0 (mg/Kg/min)= V0(mg/L/min) x EBVIBW

24

where V0 is the initial velocity of clearance in milligrams per kilogram per minute, BW is
the body weight in kilograms, and EBV is the estimated blood volume in liters, calculated
according to
EBV = (mg ICG in]ected)loxp(a)
The reciprocal of the dose (1/D) was plotted against the reciprocal of the velocity

of clearance (1N0), and a regression equation was calculated
1N0 = (1Idose)(slope) + 1Nm

In this formula, the y-intercept represented 1N max or the reciprocal of the theoretical

maximal velocity of clearance of ICG at an infinite dose of ICG (Figure 5).

Vm represents the transport maximum or the theoretical dose at which the there would be
an infinite rate of indocyanine green clearance (Hauptman, 1989). An estimate of the

hepatic blood flow (EHBF) can be obtained by the following formula
EHBF = VOIUCGlo

Where [ICG]0 is the concentration of ICG at time = 0. Mathematically this reduces to
VoIflCGlo = b exp(a)lexp(a) = b
OR
EH BF = b
The EHBF may be inaccurate because it assumes an extraction ratio of l for ICG. In
reality, it is probably closer to 0.5 or possibly even 0.3 for rats in shock. Despite this
limitation, this gives an intuitive sense for the significance of the value of the coeflicient b.
Reduced indocyanine green clearance has been found to be an indicator of early sepsis

(Wang, 1991b) despite microvascular hyperperfusion (Wang, 1991c).

 

25

 

1N0

/ \ “VI"

\
1/Km

 

 

1/dose

Figure 5. Calculation of Vm and Km from the double reciprocal plot of
l/dose vs. l/VO.

26

Assessment of microvascular blood flow

Surface blood flow was determined by laser Doppler flowmetry. This is a
measurement which is performed by placing a fiberoptic probe from the monitor on the
surface of each organ to be measured during laparotomy. The probe directs laser light
into the tissue, illuminating a volume which contains both red blood cells and stationary
tissue cells. Light energy (photons) is randomly scattered by both cell types. Photons
scattered by moving cells are Doppler shifted. Return fibers on the cable transmit the a
portion of the scattered light which is then converted into electronic signals by a
photodetector in the monitor. The results are read in arbitrary units and are processed by
the monitor (Laserflow Operator Manual).

Cytokine Measurement

Blood samples were drawn from the rat and placed in small plastic blood
containers. TNF activity was determined by assessing plasma samples for WEI-II 164
subclone 13 cytotoxicity (Eskandari, 1990). Total TNF was quantified by antigenic
assessment with TNF ELISA (Genzyne corp). IL-6 activity was determined with IL-6
dependent 7TD1 B-cell hybridoma cells (Ayala, 1991). The relative unit value of plasma
activity per milliliter of TNF and IL-6 was determined by a comparison of the curves
produced from dilution of the experimental plasma to those generated by dilution of
murine TNF standard and a human recombinant IL-6 standard.

Other Measurements

Serum GOT was measured using a kit commercially available from Sigma
company. The procedure utilizes the following reaction:

Aspartic Acid + a-ketoglutarate ——oor--> Oxalacetic Acid + Glutamic Acid
The oxalacetic or pyruvic acid formed in this reaction is reacted with 2,4-

dinitrophenylhydrazine. The color intensity of the resulting phenylhydrazones is

 

27

proportional to the transaminase activity. Colorimetric measurement was then performed

on the mixture and represents the enzyme activity (Sigma Diagnostics technical manual).
PGE2 was measured using a kit commercially available fi'om Dupont. The

technique uses a radioimmunoassay. A constant, limiting amount of antibody is added to a
mixture of the sample (or standard) and a fixed amount of radiolabeled antigen (tracer).
As the amount of antigen (PGEZ) is increased, the amount of tracer bound to antibody
decreases.
Statistical analysis

All data are expressed as mean i standard error of the mean (SEM). One-way
analysis of variance (ANOVA) and Student-Neman-Keuls tests were used and the
difl‘erences were considered significant at p value < 0.05. An ANOVA on ranks was used
if the data did not have a Gaussian distribution or the equal variance test was not passed.
The chi squared test was used for mortality analysis. SigrnastatR was used as a
computerized statistical package. All results are given as mean : SEM. *p<0.05 versus

sham, #p<0.05 vs saline.

in.-

 

RESULTS
Cardiovascular system

The heart rate of the saline treated group was significantly higher than the sham
group. The heart rate of both the GM1892 and heparin treated animals was significantly
less than the saline treated group and not significantly different than the sham group
(Figure 6). The mean arterial pressure, however, was significantly decreased in all animals
that underwent trauma-hemorrhage and resuscitation despite treatment (Figure 7). The
cardiac index as measured with indocyanine green dilution was significantly decreased in
the saline treated group compared to the sham group but was restored in both the
GM1892 and heparin treated groups (Figure 8). The calculated stroke volume was
significantly decreased in the saline group compared to the sham group but restored in the
GM1892 and heparin groups to sham levels (Figure 9). The calculated total peripheral
resistance was significantly increased in the saline group compared to the sham group but

decreased to sham levels in both the GM1892 and heparin groups (Figure 10).

28

 

29

 

520
500 e
480 -
460 4

440a

*
#
420 —
#-
4oo —
330 —
360 —
340 -
320 —
30° ‘ SHM SAL GM HEP

Figure 6. At 24 hours rats were anesthetized and a catheter placed in the
carotid artery. Heart rate (HR) was measured with a manometer as described
under "Materials and Methods". *p<0.05 versus sham, #p<0.05 vs saline.
SHM-sham group, SAL-saline group, GM-GM1892 group, HEP-heparin
group.

HR (beats per minute)

 

 

 

3O

 

120
115 -

11o —

a
105 - *
*

100 a

90 ~

85

80 J

SHM SAL GM HEP

Figure 7. At 24 hours rats were anesthetized and a catheter was placed in
the carotid artery. Mean arterial pressure (MAP) was measured using a
manometer as described under "Materials and Methods". *p<0.05 versus
sham. SHM-sham group, SAL-saline group, GM-GM1892 group, HEP-
heparin group.

MAP (mm Hg)

10
0'!
l

l

 

 

 

31

# #
* I I
SHM SAL GM HEP

Figure 8. At 24 hours after trauma-hemorrhage and resuscitation, a
fiberoptic catheter was placed in the left carotid artery to the level of the
descending thoracic aorta. Cardiac index was determined by indocyanine
green dilution as described in "Materials and Methods". *p<0.05 versus
sham, #p<0.05 vs saline. SHM-sham group, SAL-saline group, GM-
GM1892 group, HEP-heparin group.

 

32-

30—

28

1

26

l

24

l

22

1

CI (ml/min/ 100 g BW)

1

20

18

l

16

1

 

 

 

32

# #
* I I
SHM SAL GM HEP

Figure 9. Stroke volume was calculated using cardiac index and heart rate.
SV=CI/HR. ‘p<0.05 versus sham, #p<0.05 vs saline. SHM-sham group,
SAL-saline group, GM-GM1892 group, HEP-heparin group.

 

100
95 I

3838
1111

sv (ul/beat/l 00g BW)
5‘
l

-h 01 0| 0
0! O 0| 8 0|
1 1 1 1 l

 

 

 

.5
O
L

33

5.0

 

4.8 -
4.6 —
4.4 —
4.2 —
4.0 —

a
3.8 — #
3.6 — #
3.4
3.2 —
3'0 J SHM SAL GM HEP

Figure 10. Total peripheral resistance was calculated value using C] and BP
measurements. TPR = MAP/CI. *p<0.05 versus sham, #p<0.05 vs saline.
SHM-sham group, SAL-saline group, GM-GM1892 group, HEP-heparin
group.

TPR (mm Hg/ml/l 00g BW)

l

 

 

 

34

Hepatocellular function

V0 as measured with the 0.25 mg indocyanine green dose was significantly

decreased in the saline group compared to the sham group. V0 in the GM1892 and

heparin treated groups, however, was not significantly different than in the sham group
(Figure 11). The GOT level was significantly increased in the saline group compared to
the sham group. The levels in both the GM1892 and heparin groups were decreased from
the saline group and not significantly different than the sham group (Figure 12).

35

 

0.14

0.13 -

if #
0.12 —
0.11 -
0.10 .4
0.09 -I
0.08 -
0.07 — *
0.06 —
0.05 ~
0.04 —
SHM SAL GM HEP

Figure 11. V0 at 24 hours after trauma-hemorrhage and resuscitation. A

V0 (mg/kg/min)-0.25 mg ICG dose

 

 

fiberoptic catheter was placed in the left carotid artery to the level of the
descending thoracic aorta. V0 was determined by indocyanine green dilution

as described under "Materials and methods". *p<0.05 versus sham, #p<0.05
vs saline. SHM-sham group, SAL-saline group, GM-GM1892 group, HEP-
heparin group.

36

 

200
180 -
160 -
140 -
120 'l
100 -

80-

cor (SF Units/ml)

60—

40-

20-

 

 

 

 

 

SHM SAL HEP

Figure 12. Glutamic oxalacetic transaminase (GOT) at 24 hours after
trauma-hemorrhage and resuscitation. Plasma samples were obtained and
frozen. GOT was determined by colorimetry as described under "Materials
and Methods". *p<0.05 versus sham, #p<0.05 vs saline. SHM-sham group,
SAL-saline group, GM-GM1892 group, HEP-heparin group.

37

Tissue water content

Table 1 shows the tissue water content of the organs examined by weighing prior
to and after dessication. The water content of the heart was significantly increased in all
animals subjected to trauma-hemorrhage and resuscitation despite treatment. The water
content of the liver was significantly increased in saline treated animals but decreased to
sham levels in the GM1892 and heparin treated groups. The water content of the lungs
was significantly higher in the saline and GM1892 groups but the heparin group was not
significantly different that the sham group (Figure 12). The kidney water content was
significantly increased in the saline only treated group but decreased to sham levels in the
GM1892 and heparin treated groups. The water content of the spleen was significantly
increased in all the animals that underwent trauma-hemorrhage and resuscitation and
significantly less in the GM1892 group compared to the saline group. The water content
of the intestine was significantly higher in the animals that underwent trauma-hemorrhage

and resuscitation but significantly lower in the heparin group compared to the saline

group.

38

Table 1. At 24 hours after hemorrhage and resuscitation, small sections of
organs were removed. The weight of the organs was measured prior to and
after dessication as described in "Materials and Methods". Measurements are
percent. ‘p<0.05 versus Sham; #p<0.05 vs Saline.

Tissue Water Content (7.)

Sham Saline GM 1892 Heparin
HEART 76.8 i 0.57 79.7 t 0.55‘ 78.7 1 0.41‘ 78.9 i 0.67‘
LUNGS 79.5 i 1.81 82.8 i 0.73‘ 81.9 i 0.54‘ 80.0 i 0.87”
LIVER 68.6 _+. 1.71 75.5 i 0.68‘ 73.2 i 0.51"” 73.8 i 059““
KIDNEY 71.2 i 2.24 75.7 $1.045- 73.5 i 1.26 73.3 i 0.59
SPLEEN 73.7 i 0.87 79.3 i 0.738 78.1 3: 061‘“ 79.3 i 0.57‘

INTESTINE 74.3 i 0.82 76.5 i 0.983‘ 77.6 _+_ 0.61‘ 79.9 _+_ 089‘“

39

Microvascular blood flow

Table 2 shows the microvascular blood flow of the abdominal organs examined.
The blood flow of the liver was significantly decreased in the saline group and heparin
compared to the sham group. The liver blood flow was significantly higher in the
GM1892 and heparin treated groups compared to the saline group. The kidney blood
flow was significantly less in all the animals that underwent trauma-hemorrhage and
resuscitation compared to the sham group. The kidney blood flow in the GM1892 and
heparin groups was also significantly increased compared to the saline group. The splenic
blood flow and intestinal blood flow was significantly decreased in all the animals
subjected to trauma-hemorrhage and resuscitation. It was also significantly improved in

animals treated with either GM1892 or heparin.

40

Table 2. At 24 hours after hemorrhage and resuscitation, the animal was
anesthetized. A laparotomy was performed and the rrricrovascular organ
sm'face blood flow was measured using laser Doppler flowmetry as

described in "Materials and Methods". Measurements are in arbitrary units.
*p<0.05 versus Sham; #p<0.05 vs Saline.

Microvascular Blood Flow (Arbitrary Units)

Sham
LIVER 40.7 i 1.59
KIDNEY 80.4 i 1.60
SPLEEN 35.8 i 1.06

INTESTDQE 90.5 i 1.26

Saline

25.4 i 1.24‘
45.8 i 0.94“
25.3 i 0.54"
58.2 1 1.05"

GM1892
37.9 i 1.89#
60.7 i 136*”

. 28.0 i: 0.83”"

81,911.71”

Heparin

29.8 i 091"”
58.5 i 1.38"If
29.9 i 072*”
81.8 i 169*“

4]

Mediator analysis

Plasma IL-6 levels were significantly elevated in the saline group compared to the
sham group. H.—6 levels were reduced to sham levels in the GM1892 and heparin treated
groups (Figure 13). Plasma TNF levels as measured by the bioassay method were
significantly elevated in the saline group compared to the sham group. TNF levels were
also reduced to sham levels in the GM1892 and heparin treated groups (Figure 14).
Plasma TNF levels as measured by ELISA were elevated in the saline and heparin treated
groups but significantly over the sham or GM1892 treated groups (Figure 15). PGE2 was
significantly elevated in the saline group compared to the sham group but reduced to sham

levels in the GM1892 and heparin treated groups (Figure 16).

42

 

8000

6000-

 

4000 e

lL-6 (U/ml)

2000 e

 

 

 

 

 

SHM SAL HEP

Figure 13. IL-6 was measured by 7TD] cell bioassay fi'om frozen plasma
samples taken at 24 hours after trauma-hemorrhage and resuscitation as
described in "Materials and Methods". *p<0.05 versus sham, #p<0.05 vs
saline. SHM-sham group, SAL-saline group, GM-GM1892 group, HEP-
heparin group.

43

 

2N)

150—

TNF (U/ml)
é

50~

 

 

 

 

 

SHM HEP

Figure 14. TNF-a was measured by WEHI-l64 clone 13 bioassay from
frozen plasma samples taken at 24 hours after trauma-hemorrhage and
resuscitation as described in "Materials and Methods". *p<0.05 versus sham,
#p<0.05 vs saline. SHM-sham group, SAL-saline group, GM-GM1892
group, HEP-heparin group.

 

500

450 -

400 e

350 —

 

300-

250 -

200 -

TNF-alpha (pg/ml)

150 -

 

100 -

50-

 

 

 

       

HEP

SHM SAL

Figure 15. TNF as measured by enzyme linked immunoabsorbant assay
(ELI SA) at 24 hours after trauma-hemorrhage and resuscitation. The
differences between the groups were not statistically significant.

45

4000

 

3000 -

2000 -

PGE2 (pg/ml)

1000 -

 

 

   

 

 

SHM SAL HEP

Figure 16. PGEZ at 24 hours after trauma-hemorrhage and resuscitation.

’p<0.05 versus sham, #p<0.05 vs saline. SHM-sham group, SAL-saline
group, GM-GM1892 group, HEP-heparin group.

46

Mortality
There was a trend toward increased survival in GM1892 treated animals compared
with saline treated animals in a small mortality study (N=3 2). The mortality in the saline
treated group was 56% compared to 38% survival in the GM1892 treated group (Figure
17). Because of the large number of animals required to show a significant difference at

this mortality rate, this study was abandoned.

47

SURVIVAL
+ _
SALINE 7
GM1892 10

Figure 17. Mortality of saline treated animals and GM 1 892 treated animals
after trauma—hemorrhage and resuscitation. Overall mortality of the saline
treated group was 56% vs. 38% for the GM1892 treated group.

DISCUSSION

The development of intravenous fluid resuscitation was a tremendous advance in
the therapy of hemorrhagic shock. However, because fluid resuscitation alone does not
prevent many of the effects of circulating mediators in shock, pharmacologic agents have
been developed and studied in the desire to obtain this effect. Many of these agents are
available commercially and many are not. Conventional heparin was discovered to have
beneficial effects independent of its anticoagulant activity which led to the development of
modified heparins and GM1892. Previous animal studies with GM1892 have shown that
it improves hepatocellular firnction, cardiac output, and microcirculation after trauma-
hemorrhage and resuscitation acutely (up to 4 hours) in rats (Wang, 1994a). Prior to this
current study, however, it was not known whether GM1892 produced sustained beneficial
effects on organ fiinction. The mechanism of action had also not been investigated.

Because of the importance of cardiac function and hepatic function to overall
animal survival, these two organs were focused on during the course of the laboratory
work: This study showed that cardiac function is improved by administration of GM1892.
Heart rate was decreased by approximately 13% representing decreased compensation for
a depressed stroke volume. Although the MAP was not significantly different in any of
the hemorrhaged animals, there was a trend toward an increased MAP in the treated
groups supporting an improvement in cardiac fimction. The most significant cardiac
paramater that showed improvement is the cardiac index which increased by
approximately 32% in animals treated with GM1892. Similarly, by calculation, the stroke
volume increased by 57% reflecting the improvement of both the heart rate and cardiac
index. The compilation of these results show a definite improvement in cardiac function.

The data for the hepatocellular function is less clear but still convincing. The initial

rate of clearance of ICG (V0) decreased by approximately 110% in the saline group

compared to the sham group but restored in animals treated with GM 1892. Although the

48

49
V0 is dependent on both hepatocellular flinction and hepatic blood flow, the fact that the

cardiac index increased by only 32% suggests that the improvement in V0 is due to some

other factor. Most likely this other factor is an improvement in hepatocellular function.
Serum GOT, commonly used clinically to assess hepatocellular damage was decreased by
approximately 53% in animals treated with GM1892 compared to saline treated animals.
This most likely represents hepatocellular damage secondary to ischemia from the
decreased perfirsion of the organ from damaged microcirculation or directly from
circulating mediators. Liver water content was decreased in animals treated with GM1892
compared to saline treated animals. GM1892 appears to decrease the hepatocellular
damage caused by hemorrhagic shock which results in an improvement in hepatocellular
function.

The cytokine data strongly suggests cytokine release as a critical part of the
mechanism of action of GM1892. Plasma IL-6 levels were decreased by approximately
90% and plasma TNF levels were decreased by 78% in animals treated with GM1892.
Prostaglandin levels were also decreased by the administration of GM 1892. Plasma
PGE2 levels were decreased by approximately 65% in animals treated with GM1892
compared to saline treated animals. Plasma TNF as measured by ELISA was decreased in
animals treated with GM1892 compared to saline treated animals but this effect was not
statistically significant. If there had been no trend toward decreased levels TNF levels in
the GM1892 and heparin groups it would have suggested the presence of a TNF binding
protein or inhibitor. The present data with some decrease in TNF levels in the GM1892
and heparin treated group, however, lends support to an improvement in microcirculation
as a possible mechanism. Another possibility is that there is a TNF inhibitor present in
heparin but not in GM1892.

A mortality study was initiated that compared the survival of hemorrhaged animals
treated with saline compared to the survival of hemorrhaged animals treated with

GM1892. The mortality rates were different between the two groups but not significantly.

50

Although this trend would be likely to continue with more animals, such a large number of
animals (>100) would be required to show a statistical difference that this study was
abandoned.

The rat trauma-hemorrhage model used is reproducible, has a predictable outcome,
and is similar to the clinical situation of trauma followed by hemorrhage. This model also
involves multiple organ dysfirnction which is often followed by organ failure and late
mortality. The model uses unanesthetized animals to avoid the complicating effects of
anesthesia. Anesthesia lowers the metabolic demands of organs and tissues dampening the
effects of the trauma and hemorrhage. A fixed pressure mode] is also desirable because
fixed volume models depend on the state of hydration of the animal. Fixed pressure
models are physiologically similar and thus give a higher degree of reproducibility. A
nonheparinized model is also required because of the complex effects heparin has on
hemorrhaged animals as mentioned above. The trauma induced prior to hemorrhage is
significant and alone produces immune function depression (Stephan, 1987). The addition
of an initial trauma also makes the model more clinically relevant.

Crystalloid solution (RL) was used exclusively in this model for resuscitation.

Shed blood was not transfirsed. Previous investigators have concluded that the critical
systemic oxygen transport and fractional oxygen extraction are independent of hemoglobin
concentration down to hemoglobin levels of at least 40% of normal (Heusser, 1989).
Because the hemoglobin concentration in this study was approximately 50% immediately
after resuscitation and at 24 hours after resuscitation, it is unlikely that the decrease in
systemic hematocrit could cause any of the significant abnormalities that were identified.

GM1892 has been studied in the same rat model acutely after resuscitation (Wang,
1994a). In that study, the HR was found to be decreased during the entire study period.
In contrast, in the present study the HR was increased in animals that underwent trauma
and hemorrhage although less so in animals treated with GM1892. Another difference is

that the SV was not significantly reduced at 2 hours after trauma and hemorrhage

51

although it was at 24 hours in the present study. TPR was found to be decreased in
animals treated with GM1892 in both studies. The hepatocellular function was evaluated
by the Vm which was not used in the present study. Microvascular blood flow was similar
in the organs measured (liver, kidney, spleen, intestine) acutely and at 24 hours. The
protective effect of GM1892 was also similar.

Potential mechanisms of action of GM1892

The improvement in most of the measured parameters was similar between
GM1892 and conventional heparin suggesting a similar mechanism. The mechanism of
action of GM] 892 was investigated by measuring cytokine levels at 24 hours after trauma
and hemorrhage. Microvascular blood flow was increased and proinflammatory cytokines
were also decreased in those animals suggesting improvement in microcirculation as a
possible mechanism. This is supported by studies indicating that modulation of activated
neutrophils by heparin may decrease vascular injury (Greischlag, 1992). There is also an
improvement in the splenocyte response to mitogen and the capacity of splenic and
peritoneal macrophages to release IL-6 is restored in the hemorrhaged animals that receive
GM1892 or conventional heparin.

The half life of GM1892 is unknown but is most likely similar to the half life of
conventional heparin. The is no relationship between anticoagulant half-life and
concentration half-life indicating that there might be some protein binding of heparin
(Physicians Desk Reference, 1995). This does not appear likely in GM1892, however,
because the levels of TNF were decreased in both the bioassay and the ELISA. Animals
that undergo trauma-hemorrhage and resuscitation develop decreased microcirculatory
blood flow which leads to cytokine and other mediator release leading to complications
such as edema, multiple organ failure and death. Animals treated with GM1892, however,
have an improved microcirculation which attenuates the mediator release both acutely and
at 24 hours leading to decreased release of mediators, decreased complications and

possibly to improved survival (Figure 18).

/"\

Traumathemorrhage
and resuscitation

GU1892

A

Traumathemorrhage
and resuscitation

52

 

 

Massive mediator
release

A

Attenuated mediator
release

 

 

Rat dies

A

Rat lives

Figure 18. Summary of the mechanism of action of GM1892. Animals in
both groups undergo trauma-hemorrhage and resuscitation. Animals treated
with GM1892 have an attenuated mediator release leading to lower cytokine
levels at 24 hours and possibly to improved survival.

53

Other potential mechanisms may be related to heat shock proteins, protamine or
antithrombin III (AT III). Heat shock proteins may play a role, specifically, a 90-kDa
stressprotein (HSP90), which has been found to have heparin and antibody-binding
domains (Itoh, 1993). The use of protamine sulfate (a heparin antagonist) in patients has
been associated with circulatory collapse because of depressed left ventricular firnction.
Incubation with heparin before protamine addition prevents the negative effects of
protamine on myocyte function (Hird, 1994). In one study, more severely injured adult
trauma patients had low AT 111 levels at some time during hospitalization (Miller, 1994).
AT 111 is potentiated in its anticoagulation effect by heparin although as mentioned earlier,
GM1892 does not have this effect.

Other potential mechanisms of action could be similar to the mechanism of action
of heparin which has been studied to a greater extent. There is a modulation of acute
inflammation including modulation of smooth muscle cell proliferation, stabilization of
cellular membranes, and inhibition of complement activation. These could lead to
preservation of the vascular endothelium and decreased aggregation of platelets. There
are also some vascular effects which may be involved in the mechanism proposed above.
There may be an increased production of EDRF as well as modulation angiogenesis and
modulation of platelet activation and aggregation leading to improvement of red blood
cell flow to ischemic tissue in low flow situations such as shock. This protection of the

microvasculature appears to be the most probable mechanism of action.

54

Potential clinical uses

GM1892 would most likely have potential clinical uses in patients with any form of
ischemia and reperfusion injury where circulating mediators are thought to play a role.
The clinical case most similar to the model used in this study is a trauma patient with acute
hypovolemic shock secondary to hemorrhage who is resuscitated with crystalloid and has
the bleeding controlled. These patients undergo a dramatic systemic release of cytokines
resulting in multiple organ failure including ARDS and prolonged hospitalization in the
intensive care unit. GM 1 892 may attenuate the mediator release thus decreasing the risk
for multiple organ failure, sepsis and possibly decreasing the hospital stay. Other potential
uses would be for transplant patients given donor organs with prolonged warm ischemia
time, patients with peripheral vascular disease or coronary artery disease with tenuous
blood flow requiring bypass, and vascular free flaps which have had a period of ischemia.

Limitations and future studies

There are several limitations to this study. The dosing regimen used was a single 7
mg/kg dose. This was effective but the effects of multiple dosing or continuous dosing
are unknown. Other dosing regimens may provide more consistent blood levels and
provide better results. Measunnent of plasma drug levels (heparin, GM1892) would also
be helpfirl in detemrining adequate availability. In this study, a single time point (24 hours
after resuscitation) was used for measurement of TNF and IL-6. Serial cytokine
measurements may provide more insight into the pattern of cytokine production and
release in relation to trauma, hemorrhage, resuscitation, and recovery. Similarly,
continuous monitoring of any or all of the physiologic parameters may provide more
information about what organs are affected by the injury and the medication. More
detailed studies regarding the effect of GM1892 on the immune system would be helpful

to determine if the mechanism of action is similar to that of heparin. Finally, a life table

55

analysis of mortality with more frequent observations (hourly) would provide more

information and decrease the sample size required for significant results.

CONCLUSION

These results indicate that cardiovascular and hepatic functions remain depressed
at 24 hours after hemorrhage and resuscitation. Treatment with GM1892 or heparin,
however, significantly improves these functions and reduces hepatic edema. Thus,
GM1892, which unlike conventional heparin, has no significant anticoagulant activity,
appears to be a useful adjunct for maintaining cardiovascular function and hepatic integrity
following hemorrhage and resuscitation, even at 24 hours after the insult. Downregulation
of proinflammatory cytokines by GM1892 may be the mechanism responsible for the

beneficial effects of this agent following trauma-hemorrhage and resuscitation.

56

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