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Relationship between progression of atherosclerosis and coagulation measures in a randomized-controlled trial

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Relationship between progression of atherosclerosis and coagulation measures in a randomized-controlled trial

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Relationship between Progression of Atherosclerosis and Coagulation Measures in a
Randomized-Controlled Trial
by
Ronghan Li

A Thesis Presented to the
FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOSTATISTICS)

May 2022

ii

TABLE OF CONTENTS

Abstract .......................................................................................................................................... iii
Chapter 1: Introduction ................................................................................................................... 1
Chapter 2: Methods ......................................................................................................................... 2
Study design ................................................................................................................................ 2
NAPS study design ..................................................................................................................... 2
Atherosclerosis outcomes ............................................................................................................ 2
Coagulation measures ................................................................................................................. 3
Statistical analysis ....................................................................................................................... 4
Baseline group summaries. ...................................................................................................... 4
Associations of coagulation measures with atherosclerosis progression. ............................... 5
Chapter 3: Results ........................................................................................................................... 6
Demographic characteristics at baseline ..................................................................................... 6
Comparison between baseline and progression of CIMT and CAS............................................ 8
Associations with CIMT .......................................................................................................... 9
Associations with carotid artery distensibility......................................................................... 9
Associations with carotid artery compliance ......................................................................... 10
Chapter 4: Discussion ................................................................................................................... 11
References ..................................................................................................................................... 14

iii
Abstract

BACKGROUND: The Nattokinase Atherosclerosis Prevention Study (NAPS) was conducted to
test under randomized and placebo-controlled conditions the effect of nattokinase on progression
of subclinical atherosclerosis and blood measures of coagulation. While the trial analysis showed
no statistically significant effect of nattokinase on these trial outcomes, post-trial analyses
regarding associations of blood measures with atherosclerosis have not been performed.
OBJECTIVE: To determine whether coagulation measures assessed during the trial are
associated with the progression of atherosclerosis.
METHODS: Clinical data from 265 healthy individuals in NAPS were used for analysis in this
thesis. After stratified and blocked randomization, participants were followed for a median of 3
years with carotid artery intima-media thickness (CIMT) and carotid arterial stiffness (CAS)
measured at baseline (before randomization) and at every 6 months during randomized
treatment. A linear mixed effects model was used to evaluate the association between
longitudinal CIMT and CAS measures and average on-trial coagulation measures. An interaction
term of coagulation measure with a time variable (years since randomization) tested whether
each coagulation measure was associated with rate of CIMT and CAS change. Accounting for
collinearity in interaction terms at baseline and on-trial, the interaction terms of baseline and on-
trial were included in the linear mixed effects model separately.
RESULTS: Statistically significant positive longitudinal associations with CIMT progression
were noted for vWf antigen, D-dimers and ristocetin cofactor. Statistically significant negative
longitudinal associations with carotid artery distensibility progression were noted for ristocetin
cofactor, factor VIII and vWf antigen.

iv
CONCLUSIONS: Higher levels of coagulation factors are associated with increased risk of
atherosclerosis progression measured over 3 years.

1

Introduction

Natto is a traditional Japanese food that is made by fermenting soybeans with a
bacterium, Bacillus subtilis. Nattokinase is the main enzymatic substance extracted from Natto.
Nattokinase is recognized as a supplement with cardiovascular benefits. Little research has been
done to critically evaluate the effects that nattokinase has on human health to provide an
evidence base for use of nattokinase.

The Nattokinase Atherosclerosis Prevention Study (NAPS) was conducted to test under
randomized and placebo-controlled conditions the effect that Nattokinase has on atherosclerosis
progression in n = 265 healthy individuals. The primary and secondary trial atherosclerosis
progression outcomes used carotid artery B-mode ultrasonography to provide longitudinal
measures of carotid artery intima-media thickness (CIMT, primary trial outcome) and carotid
artery stiffness (CAS, secondary trial outcomes). After a median of 3 years of randomized
intervention, there were no statistically significant differences between nattokinase and placebo
groups on CIMT or CAS [1].

To evaluate other potential cardiovascular benefits of nattokinase, an array of laboratory
measures were collected from fasting blood samples drawn at repeated intervals over the trial
follow-up. Coagulation measures included prothrombin time (PT), activated partial
thromboplastin time (aPPT), fibrinogen, ristocetin cofactor, factor VIII activity, vWF antigen,
tPA antigen, PAI-1, D-dimers, and platelet aggregation with adenosine diphosphate (ADP) and
collagen type 1 and 5. While randomized allocation to nattokinase did not affect these measures

2
compared with placebo [1], association of these measures with CIMT and CAS progression was
not evaluated in the primary trial analysis.

In this thesis, NAPS data were further analyzed to assess the following question: are
coagulation measures assessed during the trial associated with the progression of atherosclerosis?
Progression of atherosclerosis was measured by change in CIMT (anatomic measure) and change
in CAS (physiologic measures).
Methods
Study design
This thesis used clinical trial data from NAPS and performed post-trial analyses using
this randomized-controlled trial.
NAPS study design
In NAPS the relationship between daily nattokinase supplementation and progression of
atherosclerosis was investigated. NAPS included 265 healthy individuals without any signs of
cardiovascular disease. Eligible participants were randomized to oral nattokinase or placebo with
a 1:1 ratio. After stratified and blocked randomization, participants were followed for a median
of 3 years with CIMT and CAS measured at baseline (before randomization) and at every 6
months during randomized treatment.
Atherosclerosis outcomes
Atherosclerosis trial outcomes in NAPS were: 1) Rate of change of intima-media
thickness of the far wall of the right distal common carotid artery (CIMT; primary trial

3
outcome); and, 2) rate of change in CAS (measured as arterial distensibility and compliance;
secondary trial outcomes) of the right distal common carotid artery determined in the same
common carotid artery segment as CIMT. CAS measurements were calculated from common
carotid artery lumen diameters measured at systole (maximum luminal diameter) and diastole
(minimal luminal diameter) and systolic and diastolic blood pressure (BP), respectively [1]. Two
CAS measures were computed as:

Coagulation measures
Coagulation measures were obtained from fasting blood samples collected at baseline and
at 1, 6, 18 and 30 months during trial follow-up. Methods of assessment of coagulation factors
are detailed elsewhere [1]. In brief prothrombin time (PT) and activated partial thromboplastin
time (aPPT) were determined by standard analytical methods by Quest Laboratories (Los
Angeles, CA) from whole blood collected into sodium-citrate tubes at room temperature [1].
Fibrinogen, ristocetin cofactor and factor VIII were determined by the University of Southern
California Clinical Laboratory from whole blood collected into sodium-citrate tubes at room
temperature and at 4° C, respectively [1].

Fibrinogen was determined by the Clauss method. Platelet aggregation with ristocetin
cofactor (von Willebrand factor (vWF) assay) was determined in plasma with a Chrono-Log
Model 700 Whole Blood/Optical Lumi Aggregometer (Havertown, PA) [1]. Factor VIII activity
was measured by a modified aPPT assay using factor VIII deficient plasma and serial dilutions

4
of participant plasma with a pooled normal plasma standard curve; values were reported as a
percentage of normal plasma [2,3]. vWF antigen was measured by ELISA with a pooled normal
plasma standard curve; results were reported as a percentage of normal plasma [2,3]. Tissue
plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) and D-dimers were
measured by ELISA in plasma [4,5]. Platelet aggregation was determined with a Chrono-Log
Model 700 Whole Blood/Optical Lumi Aggregometer (Havertown, PA) that measured platelet
function in plasma using electrical impedance (reported as Ohms) [1]. Platelet aggregation was
measured in the presence of adenosine diphosphate (ADP) and collagen types 1 and 5 [6].
Statistical analysis
Baseline group summaries.
All NAPS participants showed no signs of cardiovascular disease upon enrollment, and
were equally randomized to placebo and active nattokinase. Stratified by placebo and nattokinase
treatment groups, baseline demographic characteristics and cardiovascular risk factors of 265
randomized NAPS participants are summarized in Table 1. Medians with interquartile range
(IQR) were computed for continuous variables, including age, work hours per week, years
worked in current occupation, systolic blood pressure, diastolic blood pressure, total cholesterol,
total triglycerides, high-density lipoprotein (HDL) cholesterol and low-density lipoprotein (LDL)
cholesterol. Categorical variables, gender, marital status, race, employment status, education, and
annual income, were summarized using frequency with percentage. To test for differences of
baseline atherosclerosis risk factors between placebo and treatment groups, the normality
assumption of the distribution of both groups was examined and 2-sample t tests were conducted.
Descriptive statistics were calculated as mean and 95% confidence interval of CIMT and CAS

5
(arterial distensibility and compliance). At baseline, differences between placebo and treatment
groups on CIMT and CAS were assessed using p-values at a significance level of 0.05.
Associations of coagulation measures with atherosclerosis progression.
Normalizing transformations (primarily natural log transformation) were completed for
some laboratory measures (fibrinogen, ristocetin cofactor, factor VIII activity, PAI-1, and D-
dimers). Using a linear mixed effects model, longitudinal CIMT and CAS measures were
modeled as dependent variables with each average on-trial coagulation measure analyzed
separately as independent variables. In addition to each coagulation measure, an independent
variable of years since randomization was included in the mixed effects model; the regression
coefficient for this time variable estimated annual rate of change in CIMT or CAS over trial
follow-up. An interaction term of coagulation measure with time variable tested whether the
coagulation measure was associated with rate of CIMT and CAS change. A positive coefficient
interaction term indicated a positive association of the coagulation measure with CIMT and
CAS, and a negative coefficient interaction term indicated an inverse association. To model
repeated measures within participants, random effects at the participant level were specified for
the regression intercept (random intercept) and regression slope for time (years) variable
(random slope).

6
Results
Demographic characteristics at baseline
Table 1 shows baseline demographic characteristics, coagulation measures, and
cardiovascular disease risk factors of the 265 randomized NAPS participants with a median
(IQR) age of 65.3 (60.7, 71.9) years and BMI of 26.5 (23.7, 29.5) kg/m² , stratified by nattokinase
and placebo treatment groups. Examining categorical demographic characteristics, percent
composition of each variable was similar between randomized groups. In both groups,
approximately two-thirds of the sample was female and the majority were non-Hispanic White.
Comparing median and IQR of continuous variables, most measures (age, systolic and diastolic
blood pressure, cholesterol, triglycerides, and coagulation factors) were similarly distributed in
the two treatment groups. High-density lipoprotein cholesterol and triglycerides did not show a
statistically significant difference between medians (p = 0.28 and 0.23, respectively). Using a 2-
sample t-test to compare groups, mean and 95% confidence interval of CIMT (p = 0.51 between
groups), arterial distensibility (p = 0.06 between groups), and arterial compliance (p = 0.44
between groups) are also presented in Table 1.

7

8

Treatment group comparison on baseline and progression of CIMT and CAS
Stratified by treatment and placebo groups, Table 2 summarizes the atherosclerosis trial
outcome measures, including CIMT, arterial distensibility, and arterial compliance at baseline
and on-trial annual progression. Specifically, Table 2 presents mean and 95% confidence
intervals and p-values of CIMT and CAS for baseline and annual progression comparisons by
treatment group. P-values for baseline comparisons are greater than 0.05 level of significance
indicating no differences between treatment groups on baseline levels of the atherosclerosis
measures. P-values for interaction between treatment and time during annual progression are also

9
greater than the 0.05 level of significance, indicating no significant nattokinase effect on
atherosclerosis progression.

Associations of coagulation measures with CIMT
Table 3 summarizes beta estimates, and 95% confidence intervals and p-values for
baseline and longitudinal associations of coagulation measures with CIMT and CAS.
Longitudinal associations were tested with an interaction effect between time and coagulation
measures on CIMT and CAS in linear mixed effects models.

Statistically significant associations with baseline CIMT were noted for prothrombin time
(positive association, p = 0.04), log of factor VIII (positive association, p = 0.04), vWf antigen
(positive association, p = 0.01), and log of D-dimers (positive association, p = 0.01). Statistically
significant longitudinal associations with CIMT progression were noted for vWf antigen
(positive association, p = 0.01), log of D-dimers (positive association, p = 0.046) and log of
ristocetin cofactor (positive association, p = 0.01).
Associations of coagulation measures with carotid artery distensibility
Statistically significant associations with baseline carotid artery distensibility were noted
for log of factor VIII (negative association, p = 0.03) and vWf antigen (negative association, p =
0.01). Statistically significant longitudinal association with carotid artery distensibility
progression were noted for log of ristocetin cofactor (negative association, p = 0.02), log of
factor VIII (negative association, p = 0.049) and vWf antigen (negative association, p = 0.045).

10

Associations of coagulation measures with carotid artery compliance
No statistically significant associations of coagulation measures with either baseline
carotid artery compliance or longitudinal carotid artery compliance progression were noted.

11

Discussion

In ELITE [7], coagulation measures and carotid artery atherosclerosis and stiffness
outcomes were measured repeatedly from the same two groups of participants at common time
points at baseline and throughout the trial follow-up period. Since data obtained from the study
were longitudinal, the linear mixed effects model was selected as the statistical method to

12
evaluate associations between rates of progression of CIMT and CAS with levels of each
coagulation measure. An interaction term of coagulation measure with time (in years) on-study
tested whether each coagulation measure was associated with progression of atherosclerosis and
arterial stiffness per unit of the coagulation factor. Mixed effects modeling allows for analysis of
repeatedly measured dynamic data over time and a more precise estimation of model parameters
from the covariance matrix.

According to Gravholt et al. [8], haemostatic variables (fibrinogen, factor VIII activity,
vWF antigen, D-dimers) are positively related to CIMT. This finding is consistent with data
presented in this thesis, where NAPS data showed positive associations of CIMT and CIMT
progression with prothrombin time, factor VIII, vWf antigen, and D-dimer, and negative
associations of carotid distensibility with factor VIII, vWF antigen, and ristocetin. Therefore,
higher levels of coagulation factors are associated with increased risk of atherosclerosis.
Shorikov et al. [9] identified a negative weak correlation between carotid artery distensibility and
vWF antigen (r = -0.37, p = 0.03), which further supports results obtained in this thesis.

The three arterial wall measures analyzed in this thesis can be divided into two distinct
types of measurements. Specifically, CIMT is an anatomic measurement of structural thickness
of the inner two layers of the carotid artery to evaluate extent of carotid artery atherosclerosis.
On the other hand, carotid artery distensibility and compliance are physiological measurements
that reflect vessel wall properties assessed over the cardiac cycle. As seen in Table 3, when the
regression coefficients (both baseline and on-trial) for CIMT are small, those of carotid artery
distensibility and compliance approximate zero for the same coagulation measure. For the

13
remainder of regression coefficients, almost all showed CIMT associations in an opposite
direction to those of carotid artery distensibility and compliance for a specific coagulation
measure. Opposing directions of association reflect inverse correlation between CIMT and
carotid artery distensibility/compliance, which is consistent with the conclusion of Doyon et al.
[10] At baseline in NAPS, CIMT was inversely correlated with carotid artery compliance (r = -
0.185, p = 0.003) and showed no statistically significant correlation with carotid artery
distensibility (r = 0.003, p = 0.957). This trend is biologically plausible and reveals the
differences between anatomic and physiologic measurements.

14
References

[1] H. N. Hodis, W. J. Mack, H. J. Meiselman, V. Kalra, H. Liebman, J. Hwang-Levine, L.
Dustin, N. Kono, M. Mert, R. B. Wenby, E. Huesca, L. Rochanda, Y. Li, M. Yan, J. A. St. John,
and L. Whitfield, “Nattokinase Atherothrombotic Prevention Study: A randomized controlled
trial,” Clinical Hemorheology and Microcirculation, vol. 78, no. 4, pp. 339–353, 2021.

[2] Liebman HA, Comenzo R, Allen S, DiIorio J. A glycosaminoglycan inhibitor of
thrombin: a new mechanism for abnormal hemostatic assays in cancer. Am J Hematol.
1991;38:24-9.

[3] Rochanda L, del Zoppo GJ, Feinstein DI, Liebman HA. Approach to treatment,
characterization, and diagnosis of an acquired auto-antibody directed against prothrombin, factor
X and factor IX: a case report and review of the literature. Haemophilia. 2011;18:102-7.

[4] Liebman HA, Carfagno MK, Weitz I, Berard P, DiIorio J, Vosburgh E, Simms R.
Excessive fibrinolysis in amyloidosis resulting from elevated plasma single chain urokinase. Am
J Clin Pathol. 1992;98:534-41.

[5] Weitz IC, Razavi P, Ghods M, Rochanda L, Zwicker J, Furie B, Manly D, Mackman N,
Liebman HA. Eculizumab therapy results in rapid and sustained decreases in markers of
thrombin generation and inflammation in patients with paroxysmal nocturnal hemoglobinuria.
Thromb Res. 2012;130:361-8.

[6] Alexy T, Tucker S, Boyle S, Rowe VL, Weaver FA, Liebman HA. Heparin-PF4
antibodies are frequent following vascular surgery but are not a frequent cause of graft
thrombosis or thrombocytopenia. J Vasc Surg. 2008;48:377-81.

[7] Hodis HN, Mack WJ, Henderson VW, Shoupe D, Budoff MJ, Hwang-Levine J, Li Y,
Feng M, Dustin L, Kono N, Stanczyk FZ, Selzer RH, Azen SP for the ELITE Research Group:
Vascular effects of early versus late postmenopausal treatment with estradiol. N Engl J Med.
2016;374:1221-31.

[8] C. H. Gravholt, K. H. Mortensen, N. H. Andersen, L. Ibsen, J. Ingerslev, and B. E.
Hjerrild, “Coagulation and fibrinolytic disturbances are related to carotid intima thickness and
arterial blood pressure in Turner syndrome,” Clinical Endocrinology, vol. 76, no. 5, pp. 649–656,
2012.

[9] E. Shorikov, D. Shorikova, P. Shorikov, O. Khukhlina, “The state of carotids and the von
willebrand factor levels in patients with arterial hypertension,” ISTH Congress Abstracts.
https://abstracts.isth.org/abstract/the-state-of-carotids-and-the-von-willebrand-factor-levels-in-
patients-with-arterial-hypertension/. [Accessed: 25-Feb-2022].

15
[10] A. Doyon, D. Kracht, A. K. Bayazit, M. Deveci, A. Duzova, R. T. Krmar, M. Litwin, A.
Niemirska, B. Oguz, B. M. W. Schmidt, B. Sö zeri, U. Querfeld, A. Melk, F. Schaefer, and E.
Wühl, “Carotid artery intima-media thickness and distensibility in children and adolescents,”
Hypertension, vol. 62, no. 3, pp. 550–556, 2013.

Abstract (if available)

Abstract BACKGROUND: The Nattokinase Atherosclerosis Prevention Study (NAPS) was conducted to test under randomized and placebo-controlled conditions the effect of nattokinase on progression of subclinical atherosclerosis and blood measures of coagulation. While the trial analysis showed no statistically significant effect of nattokinase on these trial outcomes, post-trial analyses regarding associations of blood measures with atherosclerosis have not been performed.
OBJECTIVE: To determine whether coagulation measures assessed during the trial are associated with the progression of atherosclerosis.
METHODS: Clinical data from 265 healthy individuals in NAPS were used for analysis in this thesis. After stratified and blocked randomization, participants were followed for a median of 3 years with carotid artery intima-media thickness (CIMT) and carotid arterial stiffness (CAS) measured at baseline (before randomization) and at every 6 months during randomized treatment. A linear mixed effects model was used to evaluate the association between longitudinal CIMT and CAS measures and average on-trial coagulation measures. An interaction term of coagulation measure with a time variable (years since randomization) tested whether each coagulation measure was associated with rate of CIMT and CAS change. Accounting for collinearity in interaction terms at baseline and on-trial, the interaction terms of baseline and on-trial were included in the linear mixed effects model separately.
RESULTS: Statistically significant positive longitudinal associations with CIMT progression were noted for vWf antigen, D-dimers and ristocetin cofactor. Statistically significant negative longitudinal associations with carotid artery distensibility progression were noted for ristocetin cofactor, factor VIII and vWf antigen.
CONCLUSIONS: Higher levels of coagulation factors are associated with increased risk of atherosclerosis progression measured over 3 years.

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Asset Metadata

Creator Li, Ronghan (author)

Core Title Relationship between progression of atherosclerosis and coagulation measures in a randomized-controlled trial

School Keck School of Medicine

Degree Master of Science

Degree Program Biostatistics

Degree Conferral Date 2022-05

Publication Date 04/14/2022

Defense Date 05/13/2022

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag atherosclerosis,coagulation measures,linear mixed effects model,nattokinase,OAI-PMH Harvest

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Mack, Wendy Jean (committee chair), Hodis, Howard (committee member), Karim, Roksana (committee member)

Creator Email lrh9897@gmail.com,ronghanl@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC110963547

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