Confidential: For Review Only · Confidential: For Review Only 2 18 Abstract 19 Objectives – To unravel if acute fear curdles the blood as has been hypothesized since medieval 20

Confidential: For Review O

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Do bloodcurdling movies truly curdle the blood? Results

From the Fear F8ctor Crossover Trial

Journal: BMJ

Manuscript ID BMJ.2015.029359.R1

Article Type: Christmas

BMJ Journal: BMJ

Date Submitted by the Author: 09-Nov-2015

Complete List of Authors: Nemeth, Banne; Leiden University Medical Center, Clinical Epidemiology; Leiden University Medical Center, Orthopedic Surgery Scheres, Luuk; Leiden University Medical Center, Department of Clinical Epidemiology; Academic Medical Center, Department of Vascular Medicine Lijfering, Willem; Leiden University Medical Center, Clinical Epidemiology

Rosendaal, Frits; Leiden University Medical Center, Clinical Epidemiology; Leiden University Medical Center, Einthoven Laboratory for Experimental Vascular Medicine

Keywords: Fear, Venous Thrombosis, Crossover Trial, Bloodcurdling, Factor VIII

https://mc.manuscriptcentral.com/bmj

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Do bloodcurdling movies truly curdle the blood? Results from the 1

Fear F8ctor Crossover Trial 2

Banne Nemeth Medical doctor1,2

, Luuk JJ Scheres Medical doctor1,3

, Willem M Lijfering 3

Postdoctoral researcher1, Frits R Rosendaal Professor of Clinical Epidemiology 1,4 4

5

1.Department of Clinical Epidemiology, Leiden University Medical Center, 2300 RC, Leiden, The 6

Netherlands. 7

2.Department of Orthopaedic Surgery, Leiden University Medical Center, 2300 RC, Leiden, The 8

Netherlands. 9

3.Department of Vascular Medicine, Academic Medical Center, 1105 AZ, Amsterdam, The 10

Netherlands. 11

4.Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, 12

2300 RC, Leiden, The Netherlands. 13

Address for correspondence: F.R. Rosendaal, Department of Clinical Epidemiology, Leiden 14

University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands. 15

Tel.: +31 71 526 4037; Fax: +31 71 526 6994 16

Email: [email protected] 17

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Abstract 18

Objectives – To unravel if acute fear curdles the blood as has been hypothesized since medieval 19

times. 20

Design – A crossover trial in which one group of participants was exposed to a frightening (scary) 21

movie followed by a non-frightening (flat) movie. Another group of participants watched the movies 22

in opposite order. The second movie was watched at least one week after the first movie on the same 23

time of the day. 24

Setting – The movies were shown to participants who were seated in comfortable chairs, at the Home 25

Cinema of the Department of Epidemiology, which generally carries a non-frightening and relaxed 26

atmosphere. 27

Participants – 24 healthy participants, not on any medication, aged ≤30 years were recruited among 28

students, alumni and employees of the Leiden University Medical Center. 29

Interventions – A frightening movie, with potential of inducing acute fear and a non-frightening 30

(although educational), flat movie were carefully selected. Both movies had a length of approximately 31

90 minutes. 32

Main outcome measures – Primary outcome measures were markers of coagulation activity (i.e., 33

potential coagulation fear-factors): Factor VIII (FVIII:C), D-dimer, Thrombin and Antithrombin 34

complexes (TATc) and Prothrombin fragments 1+2 (F1+2). The secondary outcome was a Visual 35

Analogue Fear Scale (VAFS) on fear experienced during each movie. 36

Results – All study participants completed the study. The frightening movie was perceived more 37

frightening than the non-frightening movie (VAFS mean difference 5.4 [95%CI 4.7 – 6.1]). FVIII:C 38

levels (i.e., the difference between levels before and after the movie) were higher after the frightening 39

movie than after the non-frightening movie (mean difference of differences 11.1 IU/dL [95%CI 40

1.2;21.0]). There was no difference in effect of either type of movies on levels of TATc, D-dimer and 41

F1+2. 42

Conclusions –Acute fear affects blood coagulant factor VIII without actual thrombin formation 43

(expressed by D-dimer, F1+2, and TATc) in young and healthy adults 44

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Introduction 45

“Nothing in life is to be feared, it is only to be understood. Now is the time to understand more, so 46

that we may fear less.” Marie Curie, 1867-1934.[1] 47

For centuries the term “bloodcurdling” has been used to describe extreme frightening situations.[2] It 48

is known in several other languages of which “das blut in den Adern erstarrt”, “à vous glacer le sang”, 49

“que hiela la sangre” and “bloedstollend” are examples in German, French, Spanish and Dutch, 50

respectively. The term dates back to the concepts in medieval physiology, where it was believed that 51

the human body contained four chief fluids, blood, phlegm, bile and black bile. It was thought that 52

fear or horror would ‘run the blood cold’ or ‘curdle’ (solidify).[3] To date large parts of the complex 53

mechanism of the coagulation cascade have been unravelled and many risk factors for thrombosis 54

have been identified. However, the origin of this medieval theory has never been studied and it 55

remains unknown if fear truly induces the coagulation system and thereby curdles the blood. 56

Several studies explored the effects of physical stress on the coagulation system.[4] Chronic anxiety 57

in psychiatric patients was associated with increased levels of coagulation markers in several 58

studies.[5,6] However, chronic anxiety in psychiatric patients (with associated therapies) markedly 59

differ from a state of acute fear. In another study investigators studied coagulation markers before and 60

after bungee jumping, and reported increased coagulation activity in apparently healthy volunteers 61

after the jump.[7] However, the effects of excitement and profound physical activity are not 62

necessarily equal to those of acute fear, in particular because, as was pointed out, these individuals 63

performed these jumps voluntarily, which was seen as deviating from an ideal design.[8] To our 64

knowledge, till date no studies investigated acute fear without physical exercise and the effects on the 65

coagulation system. We hypothesize that acute fear induces activation of the coagulation system as 66

this would pose an important evolutionary benefit, which is preparing for blood loss in frightening 67

and life-threatening situations. In addition, fear may also play a role in other unexplained disturbances 68

of the coagulation cascade that might lead to venous thrombosis, as is the case in aviation.[9] 69

Here we present results from a crossover trial which was conducted to investigate whether fear is truly 70

bloodcurdling. 71

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Methods 72

Between, the 20th of July and the 11

th of August, 2015, a crossover trial was performed. Fourteen 73

healthy volunteers were first exposed to a frightening (scary) movie[10] [please add hyperlink: 74

“http://www.imdb.com/title/tt1591095/awards?ref_=tt_awd”] followed by a non-frightening (flat) 75

movie.[11] [please add hyperlink: “http://www.imdb.com/title/tt3544048/?ref_=fn_al_tt_1”]. Ten 76

other participants watched the movies in opposite order (The authors’ Christmas movie reviews are 77

provided in supplementary text 1). The second movie was watched at least one week after the first 78

movie on the same time of the day (Figure 1, study flow-chart). Participants were not on any 79

medication, aged ≤30 years and were recruited among students, alumni and employees of the Leiden 80

University. Primary outcome measures were markers of coagulation activity (i.e., potential 81

coagulation fear-factors): Factor VIII (FVIII:C), D-dimer, Thrombin and Antithrombin complexes 82

(TATc) and Prothrombin fragments 1+2 (F1+2). Blood was drawn within 15 minutes before and after 83

both movies, laboratory analyses were performed to measure coagulation markers. The secondary 84

outcome was a Visual Analogue Fear Scale (VAFS) on fear experienced during each movie 85

(supplementary figure 1). The calculated sample size based on FVIII:C and F1+2 levels was 23 86

participants (2-sided test, alpha=0.05, beta=0.9). The difference between the mean change in 87

coagulation levels (mean change of levels before and after frightening movie compared with the mean 88

change before and after non-frightening movie) were compared using a paired student t-test. Detailed 89

study methods are available in supplementary text 2. This study was approved by the medical ethics 90

committee of the Leiden University Medical Center. All participants provided written informed 91

consent. 92

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Results 93

Participants and recruitment 94

In total, 24 healthy volunteers were recruited between July 20 and August 11. Study days took place 95

on July 29, August 4, 5 and 11, all movies were shown between 19:00 and 21:00. Due to the 96

availability of participants, 14 volunteers were recruited for group A (frightening movie first), and 10 97

volunteers for group B (non-frightening movie first). Table 1 shows baseline demographics. 98

Table 1. Baseline characteristics 99

Table 1 | Study participant demographics

Demographics

Sex - male (%) 16 (67)

Age in years (mean (SD)) 25.9 (3.0)

Height in meters (mean (SD)) 1.81 (0.12)

Weight in kilograms (mean (SD)) 77.6 (11.8)

Body Mass Index 23.4 (2.5)

Smoking - Yes (%) 2 (8.3%)

Cigarettes per day

1-5 1

6-10 -

11-15 -

16-20 1

Alcohol - Yes (%) 19 (79.2)

Alcohol units per week

1-5 7

6-10 7

11-15 3

16-20 1

21-25 -

26-30 1

Coffee - Yes (%) 19 (79.2)

Cups of coffee per day

1-3 12

4-6 6

7-9 1

Favorite movie genre (number (%))

Action 7

Adventure 1

Thriller 3

Romantic Comedy 5

Science Fiction 2

Comedy 4

Fantasy 1

Drama 1

Horror movie fan - Yes (%) 5 (20.8)

Frightening movie

VAFS (mean (SD)) 5.4 (1.7)

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Movie Score (mean (SD)) 4.25 (4.3)

Already have seen movie - Yes (%) 3 (12.5)

Non-frightening movie

VAFS (mean (SD)) 0.0 (0.2)

Movie Score (mean (SD)) 5.1 (1.7)

Already have seen movie - Yes (%) 0 (0)

VAFS=Visual Analogue Fear Scale, SD=Standard deviation

100

The frightening movie was experienced as quite frightening with a mean VAFS of 5.4 (SD 1.7). Only 101

three participants had already seen this movie. The mean VAFS of the non-frightening movie was 0.0 102

(SD 0.2) and no participant had already seen this movie. More fear was experienced during the 103

frightening movie (mean difference of VAFS: 5.4 (95%CI 4.7;6.1). 104

Fear-f8ctor analyses: primary outcome measures 105

Three participants were excluded from the primary fear-f8ctor analyses. In the first two cases (group 106

B), of both participants one of the samples was visibly haemolytic as noted by the laboratory 107

technicians, and the results were considered to be unreliable. In the third case, the first two authors 108

noted (a-priori) that the participant was very tense before the first blood draw (before the non-109

frightening movie) and in attempt to relax before the second blood draw he ingested an entire family 110

pack of chocolates after the first blood draw. He suffered from vasovagal reflexes and fainted during 111

the blood draw after the first movie. At the second movie (the frightening movie) these symptoms 112

were not noted. Since the assumption for a crossover trial, i.e. both instances similar except for the 113

intervention, was clearly not met for this individual, results were regarded as unusable (FVIII:C 132 114

IU/dL before the first movie and 346 IU/dL after, and 109 IU/dL before and 123 IU/dL after the 115

frightening movie, respectively). 116

After exclusion, the remaining 21 participants were included in the main analyses. Mean FVIII:C 117

baseline levels were similar before the frightening and the non-frightening movie (mean difference -118

2.9 IU/dL (95%CI -10.1-4.2). However, the mean change in FVIII:C levels (i.e., the difference 119

between levels before and after movie) was higher after the frightening movie than for the non-120

frightening movie (mean difference 11.1 IU/dL [95%CI, 1.2;21.0]). Figure 2A shows the mean 121

change in FVIII:C levels before and after the frightening and non-frightening movie. As can been 122

appreciated from this figure, FVIII:C levels increased in 12 (57%) participants during the frightening 123

movie, and in only 3 (14%) during the educational movie. In 18 (86%) participants, FVIII:C levels 124

declined during the non-frightening movie, which happened in only 9 (43%) participants during the 125

frightening movie. 126

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Mean D-dimer levels before the frightening and non-frightening movie were similar. The mean 127

change in D-dimer levels before and after the non-frightening movie was -35.1 ng/mL (SD 63.0) and -128

31.8 (SD 109.3) before and after the frightening movie. So, no effect of acute fear on D-Dimer levels 129

was observed (Figure 2B), mean difference 3.33 ng/mL [95%CI -37.1;43.8]). 130

There was also no effect on TATc levels, mean difference of differences -0.54 µg/L (95%CI -2.5;1.4), 131

(Figure 2C). In these healthy participants, of all F1+2 measurements (before and after both movies), 132

all but two were below 200 pmol/L. The mean change in F1+2 levels (Figure 2D) before and after the 133

non-frightening movie was -10.2 pmol/L (median -1.0), the mean change after the frightening movie 134

was -23.7pmol/L (median -21). 135

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Discussion 136

In this crossover study we demonstrated that bloodcurdling fear, affects blood coagulant FVIII. We 137

successfully attempted to design a study to expose participants to undiluted and acute fear in the 138

absence of physical exertion. We assessed as coagulation fear-factor, analytes at different levels in the 139

biologic process of clotting, i.e. at the level of a candidate procoagulant factor (FVIII), thrombin 140

production (TATc, F1+2) and fibrin production (D-Dimer). An increase in one of the candidate fear-141

factors, traditionally know as factor VIII of the coagulation cascade, was observed when exposing 142

participants to a frightening movie. Increased factor VIII:C has been associated with increased venous 143

thrombosis risk.[12–14] Therefore, the mean increase in FVIII:C of 11.1 IU/dL in these healthy 144

participants exposed to acute fear could well be clinically relevant, as was previously reported that for 145

every 10 IU/dL increase in factor VIII:C levels the risk of venous thrombosis increases with 17% 146

(95%CI 7;28).[13] It is likely that for a multicausal disorder as thrombosis, other risk factors, amongst 147

which genetic risk factors, need to be present simultaneously with elevated FVIII levels. It may well 148

be, that movie-induced elevated FVIII levels increase the risk of thrombosis, but that our group of 149

healthy individuals was far too small to observe an effect on clinical disease. Therefore, we consider 150

factor VIII to be a bloodcurdling fear-factor. D-dimer, TATc and F1+2 levels did not prove 151

themselves to be a fear-factor. This indicates that while the coagulation cascade was influenced, this 152

did not lead to thrombin formation (TATc, F1+2) and subsequent fibrin formation (D-dimer). A 153

strength of this study is its cross-over design, which allows each case to be its own control. By 154

showing the movies (one week apart) on the same day and time of the week, we expect no influence 155

of circadian rhythms and lifestyle on the study results. A limitation of the study could be the 156

magnitude of fear (and thereby the bloodcurdling potential) induced by the exposure. Although some 157

participants scored a 7 or 8 on the VAFS, there is definite room for fear enhancement. However, such 158

designs might elicit ethical controversy and would be likely to be confounded by increased physical 159

activity. Secondly, we did not reach the sample size (23 participants) after exclusion of 3 participants. 160

Nevertheless, we still found a clear 11.1 IU/dL (95% CI, 1.2;21.0) increase in factor VIII:C after the 161

frightening movie. Thirdly, participants were not randomized for the order in which they watched the 162

movies. However, this depended on availability of participants and was not influenced by the 163

researchers. Finally, the VAFS was not validated which may have under- or overestimated the actual 164

experienced fear. 165

The underlying biological mechanism of acute fear resulting in increased coagulation activity is still 166

to be unravelled. However, these results are biologically and evolutionary plausible. Increased activity 167

of the coagulation system when experiencing frightening situations might pose an important 168

evolutionary benefit: preparing for blood loss in frightening situations. These findings might be 169

explained by an adrenergic response, as epinephrine has been shown to increase factor VIII 170

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concentrations.[15] Also, desmopressin, a vasopressin analogue, has been used to increase 171

coagulation activity in a wide range of bleeding diathesis.[16]) Desmopressin stimulates von 172

Willebrand factor release with increased factor VIII levels as a result.[17] A biological mechanism 173

explaining the study results might be related to these pathways. 174

Although not immediately obvious by which means our results could confer clinical benefits, a 175

broader implication of these study results is that after centuries the term: “bloodcurdling” in literature 176

is justified. 177

Conclusions 178

This study shows that acute fear truly affects blood coagulant factor VIII without actual thrombin 179

formation (expressed by D-dimer, F1+2, and TATc) in young and healthy adults. 180

“What this paper adds box” 181

1.What is already known on this subject 182

As early as in medieval times it was thought that fear induced curdling (solidifying) of the blood. 183

Several studies have reported increased coagulation profiles in times of physical or psychological 184

stress, however the effect of pure fear on the coagulation system is still unravelled. 185

2.What this study adds 186

Acute fear truly affects blood coagulation, as evidenced by a mean 11 IU/dL increase in coagulation 187

factor VIII immediately after watching a bloodcurdling movie. Since there was no effect on D-dimer, 188

TATc or F1+2 levels, we conclude that horrifying movies have a sudden effect on coagulation 189

without actual thrombin formation in young and healthy adults. In terms of the season, a truly relaxing 190

and merry Christmas, without exposure to fright, seems to be safe to prevent venous thrombosis. 191

Contributors 192

According to good epidemiological practice, the decision of who would be first and who would be 193

second author was made by randomization. All authors were involved in the study design, study 194

conduct, data analysis, and revision of the manuscript. All authors read and approved the final 195

manuscript. 196

Acknowledgements 197

We would like to thank Nine Nemeth, Maaike Hermans and Liesbeth Willems of Brilman – Tuinhof 198

de Moed for their help with blood collection. Petra Noordijk, Annelies Hoenderdos and Lejla Mahic 199

for laboratory analyses, Selina Wijbenga and Yanna van der Spek for accompanying frightened study 200

participants and Eva Rosendaal for her expertise in horrorology. This study could not have been 201

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performed without all participants, whom we would like to gratefully thank for their participation in 202

the study. 203

204

Funding 205

This study was sponsored by the Department of Clinical Epidemiology of the Leiden University 206

Medical Center. 207

Competing interest 208

All authors have completed the ICMJE uniform disclosure form at www.icmje.org/coi_disclosure.pdf 209

and declare: no support from any organisation for the submitted work; no financial relationships with 210

any organisations that might have an interest in the submitted work in the previous three years; no 211

other relationships or activities that could appear to have influenced the submitted work. 212

Ethical approval 213

All participants provided written informed consent. This study was approved by the Medical Ethical 214

Committee of the Leiden University Medical Center in Leiden, The Netherlands. 215

216

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References 217

1. Benarde MA. Our Precarious Habitat (Second Edition). W. W. Norton & Company; 1973. 218

219

2. Anderson S. Collins English Dictionary – Complete and Unabridged. 7th ed. Glasgow: 220

HarperCollins; 2005. 221

222

3. Knowles E. Oxford Dictionary of Phrase and Fable. Oxford University Press; 2005. 86-87. 223

224

4. Thrall G, Lane D, Carroll D, Lip GYH. A systematic review of the effects of acute 225

psychological stress and physical activity on haemorheology, coagulation, fibrinolysis and 226

platelet reactivity: Implications for the pathogenesis of acute coronary syndromes. Thromb 227

Res. 2007;120:819–47. 228

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5. Geiser F, Meier C, Wegener I, Imbierowicz K, Conrad R, Liedtke R, et al. Association 230

between anxiety and factors of coagulation and fibrinolysis. Psychother Psychosom. 231

2008;77:377–83. 232

233

6. Pitsavos C, Panagiotakos DB, Papageorgiou C, Tsetsekou E, Soldatos C, Stefanadis C. 234

Anxiety in relation to inflammation and coagulation markers, among healthy adults: The 235

ATTICA Study. Atherosclerosis. 2006;185:320–6. 236

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7. van Westerloo DJ, Choi G, Löwenberg EC, Truijen J, de Vos AF, Endert E, et al. Acute stress 238

elicited by bungee jumping suppresses human innate immunity. Mol Med. 2011;17:180–8. 239

240

8. Vandenbroucke JP. Bungee-jumping and design of experiments. Lancet. 1992;340(8822):800. 241

242

9. Kelman CW, Kortt M a, Becker NG, Li Z, Mathews JD, Guest CS, et al. Deep vein thrombosis 243

and air travel: record linkage study. Br Med J [Internet]. 2003;327(7423):1072. 244

245

10. Blum, J, Producer, Wan, J, Director, Whanell, L, Writer. Insidious I. Alliance Films, IM 246

Global, Haunted Movies et al. United States; 2010; 247

248

11. Kennard, D, Director. A Year in Champagne. Samuel Goldwyn Films. United States:2014. 249

250

12. Koster T, Blann AD, Briët E, Vandenbroucke JP, Rosendaal FR. Role of clotting factor VIII 251

in effect of von Willebrand factor on occurrence of deep-vein thrombosis. Lancet. 252

1995;345:152–5. 253

254

13. Kraaijenhagen RA, in’t Anker PS, Koopman MM, Reitsma PH, Prins MH, van den Ende A, et 255

al. High plasma concentration of factor VIIIc is a major risk factor for venous 256

thromboembolism. Thromb Haemost. 2000;83(1):5–9. 257

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14. Tsai AW, Cushman M, Rosamond WD, Heckbert SR, Tracy RP, Aleksic N, et al. Coagulation 259

factors, inflammation markers, and venous thromboembolism: The longitudinal investigation 260

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of thromboembolism etiology (LITE). Am J Med. 2002;113(02):636–42. 261

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15. Ingram G, Knights S, Barrow E, Dejanov I. The Rise in Factor- VIII Concentration after 263

Infusion of Adrenaline. Br J Haematol. 1968;15(3):319–27. 264

265

16. Mannucci PM, Ruggeri ZM, Pareti FI, Capitanio A. 1-Deamino-8-d-arginine vasopressin: a 266

new pharmacological approach to the management of haemophilia and von Willebrands’ 267

diseases. Lancet. 1977;1(8017):869–72. 268

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17. Mannucci PM. Desmopressin (DDAVP) in the Treatment of Bleeding Disorders: The First 20 270

Years. Blood. 1997;90(7):2515–21. 271

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18. Anand G, Shefler A, McShane T. Diagnosis and outcome of children admitted to a paediatric 273

intensive care unit with unexplained coma. Arch Dis Child. 2011;96(11):1091–1091. 274

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19. Pomp ER, Rosendaal FR, Doggen CJM. Alcohol consumptionisassociated with a decreased 276

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Figures 279

Figure 1 title 280

Flow chart study design 281

Figure 1 Legend 282

Study flowchart: 24 participants were recruited, group allocation was based on availability of the 283

study participants as movie nights were set on specific dates. Fourteen participants were exposed to 284

the frightening movie and one week later to the non-frightening movie. The remaining 10 participants 285

saw the movies in opposite order, also one week apart. Blood was drawn before the first movie (T0), 286

directly after (T1) and before (T2) and directly after (T3) the second movie. 287

Figure 2 Title 288

Mean difference in coagulation fear-factors before and after the frightening and non-frightening 289

movies. 290

Figure 2 Legend 291

2A: Absolute mean change in coagulation factor FVIII:C after exposure to a frightening and non-292

frightening movie (ordered by change in FVIII:C levels during frightening movie). Vertical bars 293

represent individual participants, the order of participants is identical in all graphs (also for 2B, 2C 294

and 2D). *-96 FVIII IU/dL difference, ** -27 FVIII IU/dL difference. 295

2B: Absolute mean change in coagulation D-dimer (after frightening and non-frightening movie). 296

2C: Absolute mean change in coagulation TATc (after frightening and non-frightening movie). 297

2D: Absolute mean change in coagulation F1+2 (after frightening and non-frightening movie). 298

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Supplements 299

Supplementary text 1, Author’s Christmas Movie Suggestions 300

Frightening movie 301

A happy family moves into a new home and all is well. At an unfortunate moment, one of the children 302

falls from a ladder and suffers from an unexplained coma. The doctors at the hospital struggle to 303

figure out the cause of the coma, but did not consider that the boy has left his physical body and is 304

lost in a different dimension (authors note: a cause not yet listed in the differential diagnosis of 305

unexplained coma in children).[18] Evil spirits start haunting the family and events soon turn grim. 306

Will the boy be rescued? How will the family stand against these evil spirits? Cuddle under your 307

blanket around the Christmas tree and find out, but be sure to keep those calf veins pumping! 308

[Please add a hyperlink this to the trailer: 309

“http://www.imdb.com/video/imdb/vi1929288729?ref_=tt_pv_vi_aiv_2”] 310

Non-frightening movie 311

This movies’ plot revolves around an esteemed wine importer who visits several famous Champagne 312

houses in the Champagne region in France. The movie focusses on the production process of 313

Champagne and the rise of the champagne industry. An absolute recommendation for all who want to 314

learn more about the world’s favourite new years’ beverage. Be sure to impress your peers during the 315

soon to come New Year’s party with the know-how from this movie! (authors note: although alcohol 316

consumption was associated with decreased venous thrombosis risk,[19] we did not expect this movie 317

to curdle the blood.) 318

Supplementary figure 1 Title 319

Visual analogue fear scale (VAFS) 320

Supplementary figure 1 Legend 321

The visual analogue fear scale (VAFS) developed by the authors to measure fear after each movie. 322

Participants indicated the amount of fear experienced during the movie, directly after watching both 323

movies. A higher number indicates more fear experienced. 324

Supplementary text 2, Detailed study methods 325

Study design 326

Between, the 20th of July and the 11th of August, 2015, a crossover trial was performed. Twenty-four 327

healthy volunteers were recruited. One group (n=14) of participants first watched a frightening movie 328

(i.e. bloodcurdling) followed by a non-frightening movie. The other group (n=10) watched the same 329

movies in opposite order. Group allocation was based on availability of the study participants as 330

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movie nights were set on specific dates. Figure 1 is a flow chart of the study design. Both movies 331

lasted for approximately 90 minutes and were watched at least one week apart, on the same time of 332

day (see flow chart). Both groups were seated in the same comfortable chairs. The participant 333

information leaflet revealed that this was a study in which the coagulation effects of sitting still and 334

watching movies was analysed. However, participants were not aware of the movie genre in advance. 335

Participants were also not informed that the actual goal was to determine if a frightening movie could 336

curdle the blood. A frightening movie[10] [please add this hyperlink: 337

“http://www.imdb.com/title/tt1591095/awards?ref_=tt_awd”] was selected carefully on fear 338

provoking potential with an external expert and was agreed upon by the two investigators. The non-339

frightening movie[11] [please add this hyperlink: 340

“http://www.imdb.com/title/tt3544048/?ref_=fn_al_tt_1”] was selected by two investigators for its 341

non-emotional provoking capacity and was (although educational), a flat (and rather dull) movie (The 342

authors’ Christmas movie reviews are provided in supplementary text 1). Before and after each 343

movie (within 15 minutes), blood was drawn through venepuncture of the cubital vein (in the 344

contralateral arm for the second draw). The movies were shown at the main meeting room, which was 345

transformed into the Home Cinema of the Department of Clinical Epidemiology of the Leiden 346

University Medical Center, which emits a non-frightening and welcoming atmosphere. Participants 347

were instructed not to consume alcohol, smoke or use any drugs on both movie days. In order to avoid 348

any confounding by superstition, no movies were shown during full moon or on Friday the 13th. All 349

participants provided written informed consent. This study was approved by the Medical Ethical 350

Committee of the Leiden University Medical Center in Leiden, The Netherlands. 351

Study population 352

Healthy volunteers, aged < 30 years were recruited among students, alumni and employees of the 353

Leiden University. Participants were not on any medication, not pregnant (or up to 6 weeks after 354

pregnancy), used no hormonal contraceptives (excluding intra-uterine devices), had no history of 355

venous thrombosis, had no major surgery or cast-immobilization of the lower extremity in the past 356

two months and did not have a malignancy or inflammatory disease. 357

Laboratory measurements 358

Measured markers of coagulation activity (e.g. potential fear-factors) were factor VIII activity in 359

IU/dL (FVIII:C) , D-dimer in ng/mL, Thrombin and Antithrombin complexes (TATc) in ug/L and 360

Prothrombin fragments 1+2 in in pmol/L (F1+2). Blood samples were drawn in vacuum tubes 361

containing 0.105M additive of Na Citrate. After blood collection, we centrifuged citrated blood at 362

2500 g for 10 minutes at 18°C within 1 hour of venepuncture. After aliquoting, samples were stored at 363

-80°C until laboratory analyses were performed. F1+2 and TATc laboratory analyses were performed 364

within one batch, all laboratory analyses were measured in duplicate. The clotting assay for FVIII:C 365

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and D-dimer was performed using the TOP analyser (Werfen Group/Instrumentation Laboratory, 366

Barcelona Spain). TATc and F1+2 were assayed via sandwich-type ELISA (Siemens, Marburg, 367

Germany). The laboratory technicians were not aware of which movie corresponded to which blood 368

sample. 369

Fear scale and questionnaire 370

After each movie a Visual Analogue Fear Scale (VAFS), designed for this specific study, 371

(supplementary figure 1) was completed by every participant. The VAFS estimates fear experienced 372

by watching the movie, ranging from no fear at all (VAFS:0) to the worst fear imaginable (VAFS:10). 373

Additionally, participants reported whether they had already seen the movie and whether they enjoyed 374

the movie on a scale from 0 (worst movie ever) to 10 (best movie ever). Finally, participants 375

completed an questionnaire on demographics, lifestyle and favourite movie genre. 376

Sample size calculation 377

Sample size was calculated for the continuous outcome variable FVIII:C (2-sided test, alpha=0.05, 378

beta=0.9). In order to observe an increase of 15 IU/dL of more (10 IU/dL leading to an increase in 379

venous thrombosis risk of around 17%) [13], a total of 23 participants were necessary (assuming that 380

the correlation between the difference in measurements at the two time points is equal to 0.7). 381

Anticipating possible drop-out of participants, we included a total of 24 participants. 382

Statistical analyses 383

Demographic and baseline data (e.g., age, sex, weight and height) were summarized as means ± 384

standard deviation (SD) or proportions. Body Mass Index (BMI) was calculated based on self-385

reported height and weight. Mean changes in the measured coagulation markers were assessed as 386

levels before and after each movie. The difference between the mean change in coagulation levels 387

(mean change of levels before and after frightening movie compared with the mean change before and 388

after non-frightening movie) were compared. A paired student t-test was used to compare the two 389

mean changes in these coagulation markers. For the VAFS we reported the mean change and 95% 390

confidence intervals (95%CI). The coagulation factor levels were similar in the group who first 391

watched the frightening movie followed by the non-frightening movie as compared with the group 392

who watched these movies in opposite order (results not shown). Therefore, a carry-over effect was 393

considered absent. For this reason, results of both study arms were pooled by type of movie exposure. 394

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Flow chart study design /

Study flowchart: 24 participants were recruited, group allocation was based on availability of the study participants as movie nights were set on specific dates. Fourteen participants were exposed to the

frightening movie and one week later to the non-frightening movie. The remaining 10 participants saw the movies in opposite order, also one week apart. Blood was drawn before the first movie (T0), directly after

(T1) and before (T2) and directly after (T3) the second movie. 292x268mm (300 x 300 DPI)

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Mean difference in coagulation fear factors before and after the frightening and non-frightening movies. /

2A: Absolute mean change in coagulation factor FVIII:C after exposure to a frightening and non-frightening movie (ordered by change in FVIII:C levels during frightening movie). Vertical bars represent individual participants, the order of participants is identical in all graphs (also for 2B, 2C and 2D). *-96 FVIII IU/dL

difference, ** -27 FVIII IU/dL difference. 2B: Absolute mean change in coagulation D-dimer (after frightening and non-frightening movie). 2C: Absolute mean change in coagulation TATc (after frightening and non-frightening movie). 2D: Absolute mean change in coagulation F1+2 (after frightening and non-frightening movie).

402x148mm (300 x 300 DPI)

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213x34mm (72 x 72 DPI)

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Confidential: For Review Only · Confidential: For Review Only 2 18 Abstract 19 Objectives – To unravel if acute fear curdles the blood as has been hypothesized since medieval 20