Disorders of the Acute Phase Response Following Trauma and Invasive Surgery

Significance: Severe injury is a leading cause of death and disability worldwide, affecting
approximately 2.8 million individuals and accounting for over 200,000 deaths annually across
the United States. Large cohort studies have demonstrated that approximately 64% of
trauma-related deaths are due to complications including thrombosis, bleeding, infection, and
organ dysfunction, and each complication corresponds with an 8% increase in risk of
mortality. While advances in critical care medicine have significantly improved the initial
survival from traumatic injuries, the proportion of morbidity and mortality from
complications experienced during traumatic convalescence has correspondingly skyrocketed.
Early in convalescence, these patients are at risk of developing life-threatening
complications instigated by trauma induced coagulopathy (TIC) and systemic inflammatory
response syndrome (SIRS). Specifically, approximately 40% of patients with severe injuries
develop TIC and approximately 70% develop SIRS. While these conditions individually increase
the risk of secondary sequelae, in concurrence, they dramatically increase the risk of
bleeding, thrombosis, infection, and multi-organ dysfunction syndrome (MODS). Later in
convalescence, patients experiencing severe injury are at risk of suffering complications
from pathologic tissue homeostasis and repair. For example, depending on the injury, 25-65%
of severely injured patients' rehabilitation and quality of life are limited by poor bone
health, including the development of osteoporosis, impaired bone healing, and/or the
development of heterotopic ossification. Together, early and late complications of
convalescence reportedly account for over $671 billion in healthcare expenditure and
disability losses yearly. Thus, there is an unmet need for improved therapeutic and
diagnostic measures for these patients to prevent or treat complications of early and late
convalescence.

The Acute Phase Response (APR): Following injury, the APR resolves the four principle
problems provoked by disruption of tissue: bleeding, pathogen invasion, tissue hypoxia, and
tissue dysfunction. After an isolated injury, e.g., femur fracture, the APR follows a
predictable and quantifiable time-course with minimal risk of complications (Figure 1A). The
survival phase contains the injury and prevents infection through hemostasis (fibrin
deposition) and acute inflammation. The later repair phase effectively removes damaged
tissue, regenerates new tissue, and restores function. Severe trauma derails the
survival-APR, provoking complications in both in early and late convalescence (Figure 1B). In
order to prevent exsanguination, severe trauma must provoke an adequate survival-APR in
proportion to the severity of the injury. However, unrestrained and prolonged activation of
coagulation and survival-inflammation lead to the development of TIC and SIRS and
significantly increasing the risk of bleeding, thrombosis, and MODS. Additionally, if a
patient persists within the survival-APR for an extended period of time, the prolonged
activation of cellular inflammation promotes disorders of tissue homeostasis, e.g.,
osteoporosis, and delays the transition to the repair-APR, stalling or preventing healing
tissue and crippling recovery in these patients. Therefore, APR complications are
mechanistically linked, that is, dysfunction of the survival-APR contributes to dysfunction
of the repair-APR. Although there are many reports suggesting potential molecular
determinants of a dysfunctional APR, the primary molecular targets driving this phenomenon
are unknown.

Severe Trauma-Provoked Changes in Plasmin Activity: Plasmin is converted from its zymogen,
plasminogen, by its activators: tissue plasminogen activator (tPA) and urokinase plasminogen
activator (uPA). While plasmin is a multifunctional protease, its canonical role is the
degradation of fibrin (fibrinolysis). While fibrinolysis occurs during the repair-APR
following an isolated injury, a severe injury provokes early hyperactivation of plasmin
(Figure 2). Fibrinolysis was first observed by anatomist Giovanni Morgagni and surgeon John
Hunter in the late 1700s during autopsies of individuals that suffered traumatic injuries.
Both Morgagni and Hunter noted that blood from these individuals strangely did not clot. It
was later determined that blood clots formed following traumatic injury or invasive surgery
spontaneously dissolved, leading to the discovery of plasmin.

The immediate and most-recognized clinical consequence of this inappropriate
hyperfibrinolysis is bleeding, as the degradation of fibrin opposes effective hemostasis. The
clinical significance of injury- induced hyperfibrinolysis was propelled by improvements in
critical care medicine that permitted not only survival of previously fatal traumatic
injuries, but also invasive, elective surgical procedures through the use of antifibrinolytic
therapeutics. Specifically, recent clinical studies in >40,000 trauma patients have
demonstrated that prevention of plasmin activation by antifibrinolytics (e.g., aminocaproic
acid (Amicar), tranexamic acid (TXA)) significantly reduced blood loss and increased survival
when administered early after injury. Thus, it has been established that inappropriate, early
plasmin activation (hyperfibrinolysis) is a significant cause of bleeding and mortality
following severe injury.

Pathologic Fibrinolysis - Hemorrhage is the Tip of the Iceberg: Both hyperfibrinolysis and
hypofibrinolysis occur following trauma and have been associated with complications
throughout convalescence indicating that plasmin's biologic role following injury is more
complex than previously understood. Indeed, since the initial investigations of plasmin's
role in bleeding, our knowledge of the biological role of plasmin has greatly expanded beyond
its role in hemostasis. Currently, it is recognized that plasmin is activated during
virtually all tissue repair, where it degrades intra- and extravascular fibrin. In addition
to fibrinolysis, plasmin also acts through non-canonical pathways to promote tissue repair
including programming and migration of macrophages and progenitor cells, growth factor
activation, and promotion of angiogenesis. Additionally, plasmin stimulates an acute
inflammatory response, promoting tissue regeneration. Thus, plasmin is essential for tissue
maintenance and repair. Specifically, our lab has extended this premise by determining that
plasmin is essential for proper bone homeostasis, as well as bone and muscle repair. Thus,
following trauma, the biological role of plasmin is not limited to intravascular activity and
bleeding. Instead, it plays well defined, albeit less understood, roles in pro- and
anti-inflammatory responses, tissue homeostasis, and repair of virtually all tissues. While
these studies would suggest that plasmin plays beneficial role in recovery following trauma,
judicious prevention of early plasmin activation is clearly beneficial in preventing life
threatening hemorrhage.

The purpose of the proposed study is to test these hypotheses through the following aims:

1. To determine if early plasmin activation following severe injury correlates with SIRS,
TIC and complications throughout convalescence in both trauma and surgical patients.

2. To determine if early plasmin activation following severe injury correlates with
plasminogen consumption and poor plasmin activity later in convalescence.

Arm 1: Level I Trauma Patients Inclusion criteria

- Any patient admitted to or treated in the VUMC Adult Trauma Unit

- Patients ages 16 and older (all included in the adult trauma unit admissions)

- Patients will be divided into sub-groups for analyses based on type of trauma (TBI,
blunt force trauma, polytrauma, etc) and severity (Level 1 vs non-level 1 trauma)
Exclusion criteria

Arm 2: Invasive Elective Surgical Patients Inclusion criteria

- Any patient admitted for an invasive elective surgery associated with high blood loss
or a high risk of vascular complications at VUMC adult hospital. Enrollment will be at
the discretion of the attending physician

- This may include, but is not limited to, orthopedic and vascular surgery patients

- Patients ages 7 years and older

Exclusion criteria

• None

Arm 3: Healthy Volunteers Inclusion criteria

- Volunteers should be male or female

- Age 18-70 years of age

- Weight greater than 110lbs

Exclusion criteria

- Chronic medical conditions such as: diabetes, hypertension, high cholesterol,
rheumatologic disorders, infections, etc.

- No history of recent traumatic injury (within the past year)

- Pregnant females or people on hormone replacement therapy

- People on any anticoagulant medication or NSAIDS