1. Background and scientific rationale Infection with the novel coronavirus SARS-CoV-2,
first identified in Wuhan, China in late 2019, has become a global pandemic
affecting over 209 countries and territories. The illness caused by SARS-CoV-2 is
classified as COVID-19. As of August 23rd, 2021, more than 212 million cases of
COVID-19 had been reported in more than 220 countries, resulting in more than 4.4
million deaths. The U.S. has by far the largest number of total cases (>38 million)
with a mortality rate of 1.67%. Although many patients may have only mild upper
respiratory symptoms, some COVID-19 patients become severely ill with respiratory
failure with risk of progression to multiple organ failure and development of
systemic coagulopathy with features similar to disseminated intravascular
coagulation (DIC).1 The pathophysiology of COVID-19-associated coagulopathy appears
to be complex and multifactorial, involving both cellular and plasmatic elements of
the hemostatic system. Development of coagulopathy is a predictor of mortality in
patients with COVID-19.2 Still, there are no direct mechanistic links established
between SARS-CoV-2 infection and coagulopathy or thrombosis.

At the University of Iowa, we led a multicenter randomized clinical trial comparing
standard prophylactic dose to intermediate dose enoxaparin in hospitalized patients
with COVID-19 (NCT04360824).3 As part of an exploratory biomarker component of this
trial, we collected blood samples from hospitalized COVID-19 patients at enrollment
and weekly for up to 30 days of hospitalization. Our pilot results, as well as
reports from other groups, demonstrate increased potential for thrombin generation
in the plasma of COVID-19 patients. In particular, in our COVID-19 patient cohort we
observed that enhanced thrombin generation potential persisted for at least 30 days
of hospitalization. We now propose to explore the mechanistic roles of activation of
blood cells (such as platelets and neutrophils), microparticles, extracellular
histones, interleukin-6 (IL6), and galectin-3 (Gal-3) as mediators of enhanced
thrombin generation in patients with COVID-19. The study design will be a
longitudinal cohort study, which will allow us to determine the time course of
enhanced thrombin generation potential in relation to clinical outcomes and changes
in markers of cellular activation in serial samples obtained from COVID-19 patients
for up to 3 years after infection with SARS-CoV-2. This study may provide clues to
why a subset of COVID-19 patients present with late thrombotic complications even
after apparent recovery from SARS-CoV-2 infection.4 Thus, this project has a strong
scientific rationale with direct clinical implications, especially given the
emergence of SARS-CoV-2 variants such as delta and omicron that may prolong the
pandemic and/or cause surges of COVID-19 in the coming months.

An ongoing question in the field relates to the comparative prothrombotic effects of
acute COVID-19 versus incidental SARS-CoV-2 infection versus acute infection with
influenza viruses. Therefore, we will include three categories of hospitalized
patients in this study: (1) acute COVID-19, (2) incidental COVID-19, and (3) acute
influenza A or B.

It is now recognized that SARS-CoV-2 infection can be associated with persistent,
relapsing, or new symptoms or other health effects occurring after acute infection,
termed "postacute sequelae of SARS-CoV-2 infection" (PASC), also known as "long
COVID" or "post-COVID condition" (PCC) or "postinfective fatigue syndrome" (PIFS).5
It is not known whether or not persistent coagulopathy is a feature of PASC.
Therefore, we will include assessment of post-COVID symptoms in our longitudinal
cohort study design, which will allow us to assess for PASC as defined by Thaweethai
et al.,2023.5

2. Objectives 2.1 Primary objectives 2.1.1 Determine the time course of enhanced
thrombin generation potential in patients with COVID-19 or influenza.

2.1.2 Test the hypothesis that activation of platelets, neutrophils, and endothelial
cells by plasma from COVID-19 patients is mediated by Gal-3, IL6, and/or histones.

2.1.3 Determine the roles of neutrophils, platelets and endothelial cells in
mediating increased thrombin generation and whether targeting IL6, Gal-3, or
histones decreases thrombin generation potential in plasma samples from patients
with COVID-19 or influenza.

2.2 Secondary objectives 2.2.1 Determine time course of changes in plasma levels of
IL6, sIL6R, H3Cit, and Gal-3 in patients with COVID-19 or influenza.

2.2.2 Determine the association of plasma levels of IL6, sIL6R, H3Cit, and Gal-3
with thrombin generation potential in patients with COVID-19 or influenza.

2.3 Exploratory objectives 2.3.1 Using mouse models of experimental venous
thrombosis, determine if infusion of plasma or microvesicles isolated from COVID-19
patient plasma potentiates thrombosis and whether neutralizing histones is
protective.

2.3.2 Determine whether plasma thrombin generation potential or plasma levels of
IL6, sIL6R, Gal-3 or H3Cit predict clinical outcomes during up to 3 years following
hospitalization with COVID-19.

3. Study design 3.1. Single-center longitudinal cohort study of hospitalized patients
with COVID-19 (laboratory-confirmed SARS-CoV-2 infection) or influenza
(laboratory-confirmed infection with influenza virus A or B).

4. Study procedures and evaluations 4.1 Screening and enrollment Potentially eligible
patients will be identified by a healthcare professional per institutional policy on
privacy. The healthcare professional will assess the eligibility of the patient by
performing a chart review which will include laboratory results. Only patients
meeting all inclusion and exclusion criteria will be asked to participate. After
obtaining verbal consent from the patient to be contacted for the study, a member of
the research staff will approach the patient to be part of the study. The research
staff will obtain informed consent from the patient or legally authorized
representative (LAR) before collecting any data and performing any procedures.

4.2 Blood sample collection Blood samples will be collected at baseline (within 24
hours of enrollment), weekly thereafter during up to 30 days of hospitalization, and
once between 3-6 months after enrollment. If the patient is discharged before the
baseline blood samples are collected, then an outpatient study visit can be
scheduled to obtain blood samples within one week of hospital discharge. Annual
telephonic follow-up and optional follow-up visits for blood sample collection will
occur at 12, 24, and 36 months after enrollment (+/- 60 days).

- Each blood sample collection will include the following (total blood volume
21.8 mL):

- Four 2.7 mL citrate tubes

- Two 3.0 mL EDTA tubes

- One 5.0 mL serum separator tube

- All blood samples are processed within one hour of collection. One tube of
citrated blood is centrifuged at 2000 g for 10 min and platelet poor plasma
(PPP) is aliquoted and snap frozen immediately for assays of cytokines and
markers of cellular activation. Samples of PPP designated for thrombin
generation assays are centrifuged again at 10,000 g for 10 min, aliquoted, and
snap frozen. Microvesicles are isolated from double centrifuged plasma by
centrifugation at 20,000 g for 30 min. A tube of citrated blood will be used to
isolate platelets. Blood collected in EDTA will be used to measure complete
blood counts and isolate leukocytes. Serum samples will be used to measure
immunoglobulins and other biomarkers.

4.3 Clinical data capture

- Clinical and demographic data will be collected by UIHC medical record review
and patient interview at the time of enrollment and each follow-up visit, and
entered into UIHC RedCap

- Data to be collected:

- Age

- Gender

- Race/ethnicity (self-reported)

- Height, weight, and body mass index

- COVID-19 vaccination history

- Medications

- Other medical conditions/past medical history

- History of thrombosis

- History of cigarette smoking

- Current symptoms Anosmia (partial or complete loss of sense of smell)
Dysguesia (altered sense of taste) Postexertional malaise (symptoms that
get worse after physical or mental effort) Chronic cough Difficulty
thinking or concentrating ("brain fog") Thirst Palpitations (fast-beating
or pounding heart) Chest pain Fatigue Decreased libido (sexual desire or
capacity) Lightheadedness or dizziness Nausea Diarrhea Abnormal movements
Fever Chills Headache Insomnia (sleep problems) Myalgia (muscle or body
aches) Sensory neuropathy (numbness or pins and needles sensation) Dyspnea
(shortness of breath) Other symptoms?

Clinical outcomes of special interest:

All-cause mortality

Thrombosis

- Arterial thrombosis, confirmed with imaging

- Venous thromboembolism, confirmed with imaging

Bleeding

- Major bleeding, defined according to ISTH criteria.6

- Minor bleeding, defined as a bleeding event that does not meet ISTH criteria
for major bleeding

5. Withdrawal or Termination Any participant who wishes to withdraw from the study may
do so at any time. If a participant chooses to withdraw from the study, no new data
about that participant will be collected for study purposes. A participant may also
withdraw authorization for the researchers to use his or her data that has already
been collected (other than data needed to keep track of the withdrawal, including
demographic data), but the participant must do this in writing to the site principal
investigator.

The study may be terminated at any time by the Principal Investigator if it is
deemed continuation of the protocol will not yield statistically or scientifically
useful data.

6. Statistical analysis Summary statistics will be provided for all laboratory
measures. Categorical measures will be presented as counts and percentages.
Continuous measure distributions will be assessed using the D'Agostino-Pearson
omnibus test and displayed as means and standard deviations or medians and IQRs,
depending on the normality of the distribution.

Tests for differences between serial samples and baseline samples will follow
Fisher's exact Student's t- or Mann-Whitney U-tests, depending on the variable type
and distribution. Analyses over time will utilize the generalized linear mixed
modeling (GLMM) framework, which accommodates repeated measures data and a variety
of outcome variable distributions. All models will include time as a predictor to
test for differences within and between groups. In addition to unadjusted
assessments, modifying factors such as age and gender will be considered for
inclusion in the models to identify the optimal predictor set. For each outcome, the
multivariate model with the smallest Akaike information criterion (AIC) will be
deemed the optimal predictor set. All tests for statistical significance of the
optimal model main effects and interactions will be conducted at alpha = 0.05 and
follow the Bonferroni adjustment based on number of model predictors to account for
multiple comparisons.

Based on our pilot data and assuming effect sizes of at least 1.54, a total of 46
subjects are needed to have >80% power when testing for differences in these
measures at alpha = 0.00625 (Bonferroni correction, 0.05/8). Anticipating technical
issues with some samples or discontinued participation of patients in the
longitudinal sample collection, we propose to recruit 60 subjects in each category
(section 4.1) to get technically adequate serial samples.

7. Study Management It is expected that the IRB will have the proper representation and
function in accordance with federally mandated regulations. The IRB should approve
the consent form and protocol. In obtaining and documenting informed consent, the
investigator should comply with the applicable regulatory requirement(s) and should
adhere to Good Clinical Practice (GCP) and to ethical principles that have their
origin in the Declaration of Helsinki. Before recruitment and enrollment onto this
study, the patient will be given a full explanation of the study and will be given
the opportunity to review the consent form. Each consent form must include all the
relevant elements currently required by the FDA Regulations and local or state
regulations. Once this essential information has been provided to the patient and
the investigator is assured that the patient understands the implications of
participating in the study, the patient will be asked to give consent to participate
in the study by signing an IRB-approved consent form. Prior to a patient's
participation in the trial, the written informed consent form should be signed and
personally dated by the patient or the patient's legally acceptable representative,
and by the person who conducted the informed consent discussion.