Platelets as Regulators of Inflammation in Cardiac Surgery

Distant organ injury is a frequent consequence of cardiovascular surgery involving
cardio-pulmonary bypass. Acute kidney injury (AKI), the most common complication, occurs in
30%-40% of cardiac surgery patients and, even with moderate damage, significantly increases
mortality and healthcare costs. Although organ injury has been linked to inflammation, the
mechanisms that initiate and propagate these events remain poorly defined. As the median age
and medical complexity of cardiac patients increases, the challenge - and opportunity - to
improve organ protection lies in identifying modifiable targets.

The investigators preliminary work provides primary evidence that activated platelets play a
novel role in the initiation of tissue injurious and inflammatory responses at the
microvascular interface. As such, platelets aggregate in close proximity to perivascular mast
cells, a cell type that can rapidly launch powerful inflammatory responses through release of
preformed effectors. Platelet activation alone is sufficient to stimulate these mast cells in
vitro while, conversely, pharmacological inhibition of platelets reduce mast cell-dependent
inflammation and end organ injury after rat circulatory arrest.

Based on compelling experimental data and evidence of human relevance, the investigators have
formed our central hypothesis that platelet activation is an important trigger of
inflammatory and tissue injurious responses after cardiopulmonary bypass through stimulation
of perivascular mast cells. The objective is to define the pathophysiologic relevance of the
platelet/mast cell interaction to end organ injury, and to identify targets for novel
strategies of organ protection in cardiac surgery patients.

Specific Aim: Define platelet activation as a risk factor for end organ injury in cardiac
surgery patients.

The investigators preliminary data strongly suggest that thrombocytopenia after cardiac
surgery is linked to AKI and increased mortality. In a prospective study of 100 patients
undergoing coronary artery bypass graft (CAGB) surgery, the investigators will define
platelet activation and consumption as the likely cause of this thrombocytopenia, and
establish the association between platelet activation in general with markers of mast cell
activation, systemic inflammation, and AKI.

Approach:

The objective of this aim is to investigate the link between perioperative platelet
activation, systemic inflammatory responses, and adverse end organ outcomes.

The investigators will perform a prospective observational study of 100 patients to
investigate the link between perioperative platelet activation, systemic inflammatory
responses, and end organ injury following cardiac surgery. Patient inclusion criteria for
this study will be based on the results from our retrospective study of 4201 patients
undergoing CABG surgery on CPB. Here, the investigators identified a significant incidence of
thrombocytopenia and an association between thrombocytopenia and increased risk for
developing postoperative AKI. Consequently, the proposed study is designed to determine
whether postoperative platelet activation contributes to a systemic inflammatory response
and, hence, has a causative effect on end organ injury.

Sample collection and processing: 15 mL of venous blood will be drawn into ACD buffer before
incision and at 10 minutes, and 6, 24, 48, and 72 hours after separation from cardiopulmonary
bypass (CPB).

Measurements: The investigators will analyze serum platelet markers by enzyme-linked
immunosorbent assay (ELISA) and the characteristic changes in membrane protein expression by
multicolor Fluorescence-activated cell sorting (FACS). Corresponding platelet counts will be
determined at each time point. Aspirin medication is given until the day of surgery and again
starting on postoperative day 2 as standard of care. Because aspirin resistance may influence
our outcomes, the investigators will measure 11-dehydrothromboxane B2 urine levels. Ensuing
subgroup analysis will establish the relevance of aspirin resistance to our observations.
Systemic inflammation will be examined by determining the plasma cytokine levels by ELISA. As
an additional arm of investigation the investigators will collect patient DNA and platelet
RNA to further examine the genomic characteristics and platelet gene expression profiles
associated with an enhanced platelet activation during surgery. This data will inform further
studies in personalized identification of risk factors for patients undergoing cardiac
surgery.

Primary outcome: AKI is a clinically relevant manifestation of end organ injury and will
serve as our primary outcome. With an incidence of 30%-40% following cardiac surgery, the
investigators expect to observe a sufficient number of cases of AKI to allow adequate
statistical analysis. The investigators will define AKI according to Kidney Disease:
Improving Global Outcomes (KDIGO) criteria and record AKI using both the proposed stages and
the change in serum creatinine values from 3 days before to 10 days after the surgery date.

Secondary outcomes: The investigators will measure cytokine expression and platelet
activation as secondary outcomes. Further, the investigators will also collect established
measures of clinical outcome such as hospital length of stay, ICU length of stay, duration of
postoperative mechanical ventilation, and major adverse cardiac events (MACE) defined as
all-cause mortality, emergent revascularization, atrial fibrillation, stroke, and non-fatal
myocardial infarction.

Patient characteristics and numbers, power-analysis, and patient Data collection: Variables
will be recorded from patient files: 1) preoperatively: age, gender, race, Society of
Thoracic Surgery risk factors, EUROScore, date of consent, and preoperative drugs; 2)
intraoperatively: number of coronary artery bypass grafts, duration of cardio-pulmonary
bypass, duration of aortic cross clamp, cumulative dose of heparin administered, blood
products, and cumulative dose of sympathomimetic and vasopressor drugs; 3) postoperatively:
timing of complications, cause and timing of death, hospital and ICU length of stay, duration
of postoperative mechanical ventilation, dose of inotropic or vasoactive support. Plate-let
count and creatinine levels, as well as anti-platelet therapy will be recorded from 3 days
be-fore to 10 days after the surgery.

Expected Outcomes. In this Aim, the investigators expect to provide new insights into the
origins of systemic inflammation and end organ injury following cardiac surgery. Our
preliminary data indicate that postoperative thrombopenia is associated with AKI and
mortality. The investigators expect to demonstrate that such thrombocytopenia occurs in a
setting of ongoing platelet activation and is associated with systemic inflammation and,
ultimately, end organ injury. Together, these investigations will lay the foundation for
novel organ protection strategies in cardiac surgery.

Statistical analysis. Descriptive comparisons will be made by using Kruskal-Wallis test or
chi-square test, as appropriate. Univariable and multivariable linear or logistic regression
analyses will be applied, as appropriate, to evaluate the association between the primary and
secondary outcomes and pre-, intra-, and postoperative variables. R2 for linear regression
analyses, odds ratios, and the corresponding 95% confidence limits for logistic regression
analyses will be re-ported.

Inclusion Criteria:

- all patients undergoing elective CABG procedures

Exclusion Criteria:

- patients with a history of:

- renal injury (creatinine > 1.5 mg/dL)

- hepatic insufficiency (liver function tests > 1.5 times the upper limit of
normal),

- severe pulmonary insufficiency (requiring home oxygen therapy),

- EF < 20%,

- IABP use preoperatively,

- liver, heart, or lung transplant