Potential Value of the Calibrated Automated Thrombogram in Patients After a Cerebral Venous Sinus Thrombosis; an Exploratory Study

Potential Value of the Calibrated Automated Thrombogram in Patients After a Cerebral Venous Sinus Thrombosis; an Exploratory Study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Potential Value of the Calibrated Automated Thrombogram in Patients After a Cerebral Venous Sinus Thrombosis; an Exploratory Study Myrthe van der Bruggen, Bram Kremers, Rene van Oerle, Robert Jan van Oostenbrugge, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-757602/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Background: Cerebral venous sinus thrombosis (CVST) is a relatively rare, but potentially lethal condition. In approximately 15% of the patients, the cause of CVST remains unclear. Conventional clotting tests such as prothrombin time and activated partial thromboplastin time are not sensitive enough to detect prothrombotic conditions nor mild haemostatic abnormalities. The calibrated automated thrombogram (CAT) is a physiological function test that might be able to detect minor aberrations in haemostasis. Therefore, we aimed to detect the presence of a prothrombotic state in patients who endured idiopathic CVST with the CAT assay. Methods: Adult patients with an idiopathic, radiologically proven CVST that had been admitted during the past 3 years were included in this study. The control group consisted of age/gender matched healthy volunteers. Exclusion criteria were known haematological disorders, malignancy (current/past) or hormonal and anticoagulant therapy recipients. We obtained venous blood samples from all participants following cessation of anticoagulation. Using the CAT assay, we determined lag time, normalized endogenous thrombin potential (ETP), ETP reduction and normalized peak height. In addition, prothrombin concentrations were determined. Results: We found no significant differences in lag time (4.7 min [4.5-4.9] vs 5.3 min [3.7-5.7], p = 0.691), normalized ETP (142% [124-148] vs 124% [88-138], p = 0.222), ETP reduction (29% [26-35] vs 28% [24-58], p >0.999), and normalized peak height (155% [153-175] vs 137 [94-154], p = 0.056) between patients and their age/gender matched controls. In addition, prothrombin concentrations did not significantly differ between patients and controls (120% [105-132] vs 127% [87-139], p >0.999. Conclusion: Reasons for absent overt hypercoagulability within this study population may be the small patient sample, long time since the event (e.g. 3 years) and avoidance of acquired risk factors like oral contraception. Given the fact that CVST is a serious condition with a more than negligible risk of venous thrombosis event recurrence, exclusion of clinically relevant hypercoagulability remains a challenging topic to further study at the acute and later time points, particularly in patients with idiopathic CVST. General Biochemistry Internal Medicine Cerebral venous sinus thrombosis (CVST) Calibrated Automated Thrombogram patients Potential value exploratory study Figures Figure 1 Figure 2 Introduction A 19-year-old female visited the emergency room with acute headache in the last 24 hours. The pain was progressive, the patient experienced nausea and vertigo, and she had vomited up to 14 times. In addition, she was both phono- and photophobic. Her medical history did not show any peculiarities and the family history was negative for thrombotic diseases. She did not suffer head trauma or infections, nor did she use any hormonal/contraceptive therapy. Magnetic resonance imaging (MRI) showed a thrombosis of the jugular vein, sigmoid sinus, right transverse sinus and the distal part of the sagittal sinus. Thrombophilia analysis did not show any of the common traits including factor V Leiden, prothrombin 20210 or inhibitor deficiencies. Despite adequate treatment with anticoagulants, the patient still regularly experienced loss of vision and headache years after the event. Cerebral venous sinus thrombosis (CVST) is a relatively rare subtype of stroke. The incidence of this disease reported in the literature is approximately 3 per 100.000 per year, affecting more women than men ( 1 , 2 ). Because of its rare occurrence the incidence of this disease might be underestimated, due to unawareness of its diagnosis. In addition, CVST is difficult to diagnose as it has a variable clinical presentation and may be challenging to confirm radiologically ( 3 ). Although CVST is a relatively rare condition, it is potentially life threatening ( 4 ). Complications include subarachnoid haemorrhage, cranial nerve palsy, epilepsy and transient ischemic attacks ( 4 – 6 ). Recent occurrences of CVST in the setting of Covid-19 vaccination, triggered interest for this disorder ( 7 ). There are several risk factors that contribute to the development of CVST. Examples are prothrombotic conditions such as protein C -, protein S -, and anti-thrombin deficiency. Furthermore, tumours, haematological disorders and the use of oral hormonal contraception are associated with CVST ( 1 , 8 , 9 ). The risk factors that contribute to the development of CVST can be identified in most patients. However, in approximately 15–20 percent of patients no cause or risk factor is identified ( 1 , 10 ). Subsequently, monitoring this patient group and estimating the risk for recurrence is rather difficult ( 11 ). In general, hypercoagulability is a key element in venous thrombosis and in addition to the mentioned thrombophilic traits, acquired factors like oral contraceptives have an important impact on coagulation through an acquired resistance against activated protein C ( 12 ). The intrinsic coagulation properties can be assessed through the calibrated automated thrombogram (CAT). The CAT, developed by Hemker and coworkers, is a semi-automated thrombin generation technique, which provides the ability to monitor thrombin concentrations in time, as the substrate for thrombin is fluorescently labeled ( 13 , 14 ). The CAT method is currently well-accepted as a research tool and has proven to be useful in several different domains such as platelet-plasma interactions, detection and quantification of thrombotic/bleeding tendency, and control of pro-coagulant and antithrombotic therapy ( 12 , 15 – 17 ). During a curiosity driven exploration of the CAT data in consecutive patients referred to the vascular outpatient clinic, we observed three patients who suffered from idiopathic CVST and had substantial elevations in thrombin generation (i.e. mean endogenous thrombin potential (ETP) 190%, mean normalized peak 352%) without any reasonable explanation. Based on this unpublished observation we designed the present study to investigate whether abnormal CAT responses would indeed be a consistent finding in patients who suffered a CVST. Methods Adult patients who endured CVST without known cause or risk factors were included in this patient study. We searched hospital records for CVST patients between January 1st 2012 and May 24th 2017 using the Dutch financial coding system for hospital care (DBC-codes). There is no specific code for CVST or for cerebral venous thrombosis. Therefore, all records of patients assigned to the code “ischemic stroke”, “haemorrhagic stroke”, “headache” and “not other specified” were screened. Based on this search strategy we identified 29 CVST patients. Next, patients under 18 years of age, patients with known coagulation disorders, malignancy (in the past), using hormonal contraception or other hormonal therapy, or anticoagulants and patients who are mentally disabled, were excluded from this study. Blood plasma from healthy age and gender matched volunteers was used as reference material. Healthy volunteers were recruited through advertisements at the faculty of health, medicine and life sciences at Maastricht University. The same exclusion criteria as with patients applied. All subjects who were eligible for inclusion in this study underwent a venepuncture and filled in a questionnaire with regard to thrombotic risk factors. This was done to place outcome measures in a clinical perspective. All subjects were informed and provided written consent. The study was performed at Maastricht University Medical Centre (MUMC+) and approved by the local medical ethical committee (Medical ethical committee MUMC, approval number; NL 63775.068.17). Blood collection and storage Four 9mL tubes with 3.2% trisodium citrate were collected through antecubital venipuncture. The blood was processed to platelet poor plasma (PPP) within 1 hour after collection via previously described methods ( 18 , 19 ). PPP was stored at -80⁰C until analysis ( 31 ). All samples were analyzed at one time point to prevent repeated freeze-thaw cycles. Plasma from healthy volunteers was processed and stored in the same manner, with the same number of freeze-thaw cycles as plasma from patients. Markers of coagulation Thrombin generation: The coagulation potential in plasma was assessed using the CAT assay (Thrombinoscope BV, Maastricht, the Netherlands). Within this method, low-affinity fluorogenic substrate for thrombin (Z-Gly-Gly-Arg-AMC; Bachem, Bubendorf, Switzerland) is added to allow continuous monitoring of thrombin formation. For each measurement, 80 µL of human PPP was added to 20 µL of fluorogenic substrate, 20 µL of trigger reagent and calcium chloride, as previous reported ( 20 , 21 ). The CAT assay was performed with and without the presence of soluble thrombomodulin (TM; Asahi Kasei Pharma Corporation, Tagata, Japan), to enable protein C depend testing ( 12 ). TG curves were calculated using Thrombinoscope software (Thrombinoscope, Maastricht, The Netherlands). Analysis resulted in four main outcome parameters: 1. Lag time; the time until clotting occurs. 2. ETP; the total amount of thrombin formed during the measurement, i.e. the area under the curve. 3. Peak height; maximum amount of thrombin generation. 4. Peak reduction; time needed for clot degradation ( 20 ). Prothrombin levels: Being an important determinant of the CAT, prothrombin was measured with a one stage FII assay on a Siemens BCSxp instrument according to the manufacturer’s instructions. Statistical analysis Baseline characteristics were collected and tabulated. Differences in thrombin generation outcome measures between patients and controls were analyzed using the Mann-Whitney U test (nonparametric) because of the small sample size. Results are shown as the median and 25th -75th percentile. P-values < 0.05 were considered statistically significant. All analyses were performed using GraphPad Prism version 7 for Windows, GraphPad Software, La Jolla California USA, www.graphpad.com . Results Baseline characteristics Eleven patients were eligible for participation in the study. Four patients were not interested in participating. From the remaining seven patients, one patient did not appear at the appointment and one patient used anticoagulant drugs despite the screening efforts. Consequently, 5 patients and 5 controls were enrolled in the study. A flow-chart of the inclusion- and exclusion process can be found in Fig. 1. Patient characteristics of all study participants are shown in Table 1 . No differences in key risk factors and relevant medication between patients and controls were observed. Table 1 Baseline characteristics Patients Controls Female gender (%) 100% 100% Age in years (mean) 42 (± 12) 46 (± 13) Years after event (mean) 2,5 Antiplatelet medication 0 0 Anti-inflammatory drugs 0 1 Alcohol consumption (U/week) 1 2 Smoking 0 0 Markers of coagulation 1.1 No significant differences in thrombin generation were observed The CAT thrombin generation assay was performed and lag time, ETP, ETP reduction and peak height were assessed [Table 2 ]. There was no significant difference in lag time between patients and controls ( p = 0.691) [figure 2A]. Although normalized peak height and ETP were both higher in patients than controls, neither difference was statistically significant ( p = 0.056, p = 0.222 respectively) (Table 2 and Fig. 2B/C). ETP reduction, which is the difference in ETP determined with and without presence of TM, was similar between the groups [Table 2 and Fig. 2D]. Table 2 Thrombin generation outcome measures depicted shown as median [25–75 percentiles]. No significant differences were observed. ETP = endogenous thrombin potential. Patients Controls P-value Lag time (min) 4.7 [4.5–4.9] 5.3 [3.7–5.7] 0.691 Normalized ETP (%) 142 [124–148] 124 [88–138] 0.222 ETP reduction (%) 29 [26–35] 28 [24–58] > 0.999 Normalized Peak Height (%) 155 [153–175] 137.4 [94–154] 0.056 Prothrombin concentration (%) 120 [105–132] 127 [87–139] > 0.999 1.2 No significant differences in prothrombin concentration were observed Prothrombin was tested as it is known to be a main determinant of peak height and ETP (22-24). No significant difference in prothrombin concentration was observed [table 2/figure 2E]. Discussion The present study assessed the presence of unexplained hypercoagulability in patients well beyond the acute phase of a CVST. We analysed thrombin generation by CAT based on a previous finding of elevated TG levels in 3 patients with CVST seen at the outpatient clinic. Two of the original 3 patients were included in the present study, the third patient was on rivaroxaban and could for that reason not be studied. Thrombin generation via de CAT method was performed in order to assess hypercoagulability. The first outcome parameter we considered was lag time. As expected, lag time did not significantly differ between patients and controls, as idiopathic hypercoagulability is mainly reflected by an increase in ETP and peak height ( 23 , 25 ). Subsequently we determined ETP, ETP reduction and peak height. ETP did not significantly differ between patients and controls. We tested ETP reduction by adding TM. Binding of thrombin to TM activates Protein C, a potent anticoagulant factor ( 13 , 26 ). We did not observe a significant difference in ETP reduction. In addition, peak height of the thrombin generation curve was determined. Although there is a visual difference between the two groups (Fig. 2B), this did not reach statistical significance, probably due to the small sample size. Based on a previous study which showed increased ETP and peak height in patients after deep venous thrombosis up till two years after the event ( 24 ), we expected to detect a similar difference in our study population. Other studies do support the findings of increased ETP and peak height after a thrombotic event ( 24 ). The clinical relevance would be found in the ability to predict recurrent thrombotic events. However, results regarding the predictive value of the thrombin generation assay on development of a secondary event (venous thrombotic event) vary widely ( 27 – 32 ). It should be mentioned that some studies used whole blood, others used PPP or platelet rich plasma, which makes it difficult to compare the outcomes. Nevertheless, we can conclude that the role of thrombin generation as a predictor is still uncertain. Patients with a first episode of CVST, a potentially devastating condition, are usually treated with anticoagulation for a limited time period, ranging from 3 to 6 months. This time is defined, considering the risk of recurrent disease after cessation of anticoagulation as low. However, this risk is not negligible and incidences ranging from 3 to 18 percent – depending on the risk factors – for development of recurrence CVST and/or other venous thrombotic events (VTE) have been reported ( 1 , 10 , 11 ). Literature suggests that the risk of recurrence is highest up till one till two year(s) after the event ( 11 , 33 ). Therefore, characterizing thrombosis risk after stopping anticoagulation may be useful. In all patients with CVST, avoidable risk factors like oral contraceptives are typically recorded and eliminated, whereas in younger individual’s variable thrombophilia screening is done. Since thrombin generation might have been a convenient single test for detecting thrombophilia, we focused on its use in this population. Study limitations Limitations of this study include the small number of participants remaining after an extensive search and selection process. CVST is a relatively rare disease and we selected an even smaller group by only including idiopathic CVST patients. The direction of some of the TG comparisons showed a trend towards hypercoagulability in the patients and it may be assumed that in a larger population true differences may emerge. Furthermore, we excluded patients with a severe course of the disease, as we excluded disabled/incapacitated subjects. This might have led to selection bias in the current population. Finally, the time after the event might be too long to be able to detect hypercoagulability and in case of risk factors like oral contraceptives, these were eliminated after the first event. We included patients after three years on average. It might simply be that the hypercoagulable state tends to normalize after this time interval. Conclusions In this pilot study we did not find any significant aberration in haemostasis between patients that suffered from CVST in the past, and age/gender machted controls. This is potentially due to a small sample size and relatively long follow-up time. Given the fact that CVST is a serious condition with a more than negligible risk of (VTE) recurrence, exclusion of clinically relevant hypercoagulability remains a challenging topic to further study at the acute and later time points, particularly in patients with idiopathic CVST. List Of Abbreviations CAT Calibrated automated thrombogram CVST Cerebral venous sinus thrombosis DBC Diagnosis-treatment combinations ETP Endogenous thrombin potential MUMC Maastricht university medical centre PPP Platelet poor plasma TM Thrombomodulin VTE Venous thrombotic event Declarations Ethics approval and consent to participate The study was performed at the MUMC and approved by the local medical ethical committee (Medical ethical committee MUMC, approval number; NL 63775.068.17 All participants gave written informed consent. Consent for publication Not applicable. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding No specific funding was obtained for this study. Authors' contributions MB, RO, RJO, and HT conceived and designed the study. MB, BK and RO performed the measurements. MB, RO and HT analysed the data and interpreted the results. MB drafted the work. MB, BK, RO, RJO, and HT critically edited and revised the manuscript. MB, BK, RO, RJO, and HT have approved the final version of the manuscript and agree to be accountable for their contributions. Acknowledgements Not applicable. References Weimar C. Diagnosis and treatment of cerebral venous and sinus thrombosis. Curr Neurol Neurosci Rep. 2014;14(1):417. Silvis SM, de Sousa DA, Ferro JM, Coutinho JM. Cerebral venous thrombosis. Nat Rev Neurol. 2017;13(9):555-65. 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Cite Share Download PDF Status: Under Review Version 1 posted Review # 1 received at journal 23 Aug, 2021 Editorial decision: Minor revision 23 Aug, 2021 Review # 2 received at journal 14 Aug, 2021 Reviewer # 2 agreed at journal 10 Aug, 2021 Reviews received at journal 10 Aug, 2021 Reviewer # 1 agreed at journal 09 Aug, 2021 Reviewers invited by journal 01 Aug, 2021 Editor assigned by journal 30 Jul, 2021 Submission checks completed at journal 27 Jul, 2021 Editor invited by journal 27 Jul, 2021 First submitted to journal 27 Jul, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-757602","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":43148409,"identity":"4e0aae03-b693-4d59-bfcd-bef8f88a492e","order_by":0,"name":"Myrthe van der Bruggen","email":"","orcid":"","institution":"Universiteit Maastricht CARIM School for Cardiovascular Diseases: Universiteit Maastricht Cardiovascular Research Institute Maastricht","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Myrthe","middleName":"van der","lastName":"Bruggen","suffix":""},{"id":43148410,"identity":"e47dd5f3-40e3-40f6-9912-8425daa432fc","order_by":1,"name":"Bram Kremers","email":"","orcid":"","institution":"CARIM: Universiteit Maastricht Cardiovascular Research Institute Maastricht","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bram","middleName":"","lastName":"Kremers","suffix":""},{"id":43148411,"identity":"966e8677-e4a6-40b7-a2a5-c49265528d46","order_by":2,"name":"Rene van Oerle","email":"","orcid":"","institution":"CARIM: Universiteit Maastricht Cardiovascular Research Institute Maastricht","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rene","middleName":"van","lastName":"Oerle","suffix":""},{"id":43148412,"identity":"f74ac651-3917-4f1c-8b69-641941632ce9","order_by":3,"name":"Robert Jan van Oostenbrugge","email":"","orcid":"","institution":"Department of Neurology, Maastricht University Medical Center, Maastricht, The Netherlands","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Robert","middleName":"Jan van","lastName":"Oostenbrugge","suffix":""},{"id":43148413,"identity":"31f9afdd-1a9d-4886-83b2-e53d864787c6","order_by":4,"name":"Hugo ten Cate","email":"data:image/png;base64,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","orcid":"","institution":"Department of Biochemistry, CARIM School for Cardiovascular Diseases, Maastricht University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Hugo","middleName":"ten","lastName":"Cate","suffix":""}],"badges":[],"createdAt":"2021-07-28 10:55:57","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-757602/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-757602/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":12084327,"identity":"07d5fdc8-8370-4db9-ae50-7326e6fecdfc","added_by":"auto","created_at":"2021-08-03 21:12:09","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":56290,"visible":true,"origin":"","legend":"Flowchart inclusion. CVST=Cerebral venous sinus thrombosis. MUMC=Maastricht University Medical Centre.","description":"","filename":"submissionfileCVSTstudyFigure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-757602/v1/333bca2b4daf293fedb4ac0f.jpg"},{"id":12084326,"identity":"fb7a2a88-1b9b-49f6-b0db-5748ddb60053","added_by":"auto","created_at":"2021-08-03 21:12:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":533206,"visible":true,"origin":"","legend":"Thrombin generation and prothrombin measurements in cerebral venous sinus thrombosis (CVST) patients and healthy controls. (A) Lag time (B) Normalized peak height. (C) Normalized endogenous thrombin potential (ETP). (D) ETP reduction (E) Prothrombin concentration. ","description":"","filename":"SubmissionfileCVSTstudyFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-757602/v1/75714b78ba9d635ab855856f.png"},{"id":13707226,"identity":"b74dc34a-e961-4d80-aef3-ec004b72ee38","added_by":"auto","created_at":"2021-09-17 14:01:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":415291,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-757602/v1/9d86d00f-4384-4790-99de-8db984d8ef80.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003ePotential Value of the Calibrated Automated Thrombogram in Patients After a Cerebral Venous Sinus Thrombosis; an Exploratory Study\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eA 19-year-old female visited the emergency room with acute headache in the last 24 hours. The pain was progressive, the patient experienced nausea and vertigo, and she had vomited up to 14 times. In addition, she was both phono- and photophobic. Her medical history did not show any peculiarities and the family history was negative for thrombotic diseases. She did not suffer head trauma or infections, nor did she use any hormonal/contraceptive therapy. Magnetic resonance imaging (MRI) showed a thrombosis of the jugular vein, sigmoid sinus, right transverse sinus and the distal part of the sagittal sinus. Thrombophilia analysis did not show any of the common traits including factor V Leiden, prothrombin 20210 or inhibitor deficiencies. Despite adequate treatment with anticoagulants, the patient still regularly experienced loss of vision and headache years after the event.\u003c/p\u003e \u003cp\u003eCerebral venous sinus thrombosis (CVST) is a relatively rare subtype of stroke. The incidence of this disease reported in the literature is approximately 3 per 100.000 per year, affecting more women than men (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Because of its rare occurrence the incidence of this disease might be underestimated, due to unawareness of its diagnosis. In addition, CVST is difficult to diagnose as it has a variable clinical presentation and may be challenging to confirm radiologically (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Although CVST is a relatively rare condition, it is potentially life threatening (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Complications include subarachnoid haemorrhage, cranial nerve palsy, epilepsy and transient ischemic attacks (\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Recent occurrences of CVST in the setting of Covid-19 vaccination, triggered interest for this disorder (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). There are several risk factors that contribute to the development of CVST. Examples are prothrombotic conditions such as protein C -, protein S -, and anti-thrombin deficiency. Furthermore, tumours, haematological disorders and the use of oral hormonal contraception are associated with CVST (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). The risk factors that contribute to the development of CVST can be identified in most patients. However, in approximately 15\u0026ndash;20 percent of patients no cause or risk factor is identified (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Subsequently, monitoring this patient group and estimating the risk for recurrence is rather difficult (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn general, hypercoagulability is a key element in venous thrombosis and in addition to the mentioned thrombophilic traits, acquired factors like oral contraceptives have an important impact on coagulation through an acquired resistance against activated protein C (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). The intrinsic coagulation properties can be assessed through the calibrated automated thrombogram (CAT). The CAT, developed by Hemker and coworkers, is a semi-automated thrombin generation technique, which provides the ability to monitor thrombin concentrations in time, as the substrate for thrombin is fluorescently labeled (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). The CAT method is currently well-accepted as a research tool and has proven to be useful in several different domains such as platelet-plasma interactions, detection and quantification of thrombotic/bleeding tendency, and control of pro-coagulant and antithrombotic therapy (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). During a curiosity driven exploration of the CAT data in consecutive patients referred to the vascular outpatient clinic, we observed three patients who suffered from idiopathic CVST and had substantial elevations in thrombin generation (i.e. mean endogenous thrombin potential (ETP) 190%, mean normalized peak 352%) without any reasonable explanation.\u003c/p\u003e \u003cp\u003eBased on this unpublished observation we designed the present study to investigate whether abnormal CAT responses would indeed be a consistent finding in patients who suffered a CVST.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eAdult patients who endured CVST without known cause or risk factors were included in this patient study. We searched hospital records for CVST patients between January 1st 2012 and May 24th 2017 using the Dutch financial coding system for hospital care (DBC-codes). There is no specific code for CVST or for cerebral venous thrombosis. Therefore, all records of patients assigned to the code \u0026ldquo;ischemic stroke\u0026rdquo;, \u0026ldquo;haemorrhagic stroke\u0026rdquo;, \u0026ldquo;headache\u0026rdquo; and \u0026ldquo;not other specified\u0026rdquo; were screened. Based on this search strategy we identified 29 CVST patients. Next, patients under 18 years of age, patients with known coagulation disorders, malignancy (in the past), using hormonal contraception or other hormonal therapy, or anticoagulants and patients who are mentally disabled, were excluded from this study.\u003c/p\u003e\n\u003cp\u003eBlood plasma from healthy age and gender matched volunteers was used as reference material. Healthy volunteers were recruited through advertisements at the faculty of health, medicine and life sciences at Maastricht University. The same exclusion criteria as with patients applied.\u003c/p\u003e\n\u003cp\u003eAll subjects who were eligible for inclusion in this study underwent a venepuncture and filled in a questionnaire with regard to thrombotic risk factors. This was done to place outcome measures in a clinical perspective.\u003c/p\u003e\n\u003cp\u003eAll subjects were informed and provided written consent.\u003c/p\u003e\n\u003cp\u003eThe study was performed at Maastricht University Medical Centre (MUMC+) and approved by the local medical ethical committee (Medical ethical committee MUMC, approval number; NL 63775.068.17).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBlood collection and storage\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFour 9mL tubes with 3.2% trisodium citrate were collected through antecubital venipuncture. The blood was processed to platelet poor plasma (PPP) within 1 hour after collection via previously described methods (\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e). PPP was stored at -80⁰C until analysis (\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e). All samples were analyzed at one time point to prevent repeated freeze-thaw cycles. Plasma from healthy volunteers was processed and stored in the same manner, with the same number of freeze-thaw cycles as plasma from patients.\u003c/p\u003e\n\u003cdiv class=\"Section2\" id=\"Sec5\"\u003e\n \u003cp\u003e\u003cstrong\u003eMarkers of coagulation\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThrombin generation:\u003c/p\u003e\n \u003cp\u003eThe coagulation potential in plasma was assessed using the CAT assay (Thrombinoscope BV, Maastricht, the Netherlands). Within this method, low-affinity fluorogenic substrate for thrombin (Z-Gly-Gly-Arg-AMC; Bachem, Bubendorf, Switzerland) is added to allow continuous monitoring of thrombin formation. For each measurement, 80 \u0026micro;L of human PPP was added to 20 \u0026micro;L of fluorogenic substrate, 20 \u0026micro;L of trigger reagent and calcium chloride, as previous reported (\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e). The CAT assay was performed with and without the presence of soluble thrombomodulin (TM; Asahi Kasei Pharma Corporation, Tagata, Japan), to enable protein C depend testing (\u003cspan class=\"CitationRef\"\u003e12\u003c/span\u003e). TG curves were calculated using Thrombinoscope software (Thrombinoscope, Maastricht, The Netherlands). Analysis resulted in four main outcome parameters: 1. Lag time; the time until clotting occurs. 2. ETP; the total amount of thrombin formed during the measurement, i.e. the area under the curve. 3. Peak height; maximum amount of thrombin generation. 4. Peak reduction; time needed for clot degradation (\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec6\"\u003e\n \u003cp\u003eProthrombin levels:\u003c/p\u003e\n \u003cp\u003eBeing an important determinant of the CAT, prothrombin was measured with a one stage FII assay on a Siemens BCSxp instrument according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec7\"\u003e\n \u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eBaseline characteristics were collected and tabulated. Differences in thrombin generation outcome measures between patients and controls were analyzed using the Mann-Whitney U test (nonparametric) because of the small sample size. Results are shown as the median and 25th -75th percentile.\u003c/p\u003e\n \u003cp\u003eP-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant. All analyses were performed using GraphPad Prism version 7 for Windows, GraphPad Software, La Jolla California USA, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.graphpad.com\" target=\"_blank\"\u003ewww.graphpad.com\u003c/a\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv class=\"Section2\" id=\"Sec9\"\u003e\n \u003cp\u003e\u003cstrong\u003eBaseline characteristics\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eEleven patients were eligible for participation in the study. Four patients were not interested in participating. From the remaining seven patients, one patient did not appear at the appointment and one patient used anticoagulant drugs despite the screening efforts. Consequently, 5 patients and 5 controls were enrolled in the study. A flow-chart of the inclusion- and exclusion process can be found in Fig.\u0026nbsp;1.\u003c/p\u003e\n \u003cp\u003ePatient characteristics of all study participants are shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. No differences in key risk factors and relevant medication between patients and controls were observed.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable border=\"1\" id=\"Tab1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eBaseline characteristics\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePatients\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eControls\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFemale gender (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAge in years (mean)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42 (\u0026plusmn;\u0026thinsp;12)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46 (\u0026plusmn;\u0026thinsp;13)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYears after event (mean)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2,5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAntiplatelet medication\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAnti-inflammatory drugs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAlcohol consumption (U/week)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSmoking\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eMarkers of coagulation\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec11\"\u003e\n \u003cp\u003e1.1 No significant differences in thrombin generation were observed\u003c/p\u003e\n \u003cp\u003eThe CAT thrombin generation assay was performed and lag time, ETP, ETP reduction and peak height were assessed [Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e]. There was no significant difference in lag time between patients and controls (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.691) [figure 2A]. Although normalized peak height and ETP were both higher in patients than controls, neither difference was statistically significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.056, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.222 respectively) (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig. 2B/C). ETP reduction, which is the difference in ETP determined with and without presence of TM, was similar between the groups [Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig. 2D].\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable border=\"1\" id=\"Tab2\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eThrombin generation outcome measures depicted shown as median [25\u0026ndash;75 percentiles]. No significant differences were observed. ETP\u0026thinsp;=\u0026thinsp;endogenous thrombin potential.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePatients\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eControls\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eP-value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLag time (min)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.7 [4.5\u0026ndash;4.9]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.3 [3.7\u0026ndash;5.7]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.691\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNormalized ETP (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e142 [124\u0026ndash;148]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e124 [88\u0026ndash;138]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.222\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eETP reduction (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29 [26\u0026ndash;35]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28 [24\u0026ndash;58]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;0.999\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNormalized Peak Height (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e155 [153\u0026ndash;175]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e137.4 [94\u0026ndash;154]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.056\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eProthrombin concentration (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e120 [105\u0026ndash;132]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e127 [87\u0026ndash;139]\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;0.999\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec12\"\u003e\n \u003cp\u003e1.2 No significant differences in prothrombin concentration were observed\u003c/p\u003e\n \u003cp\u003eProthrombin was tested as it is known to be a main determinant of peak height and ETP (22-24). No significant difference in prothrombin concentration was observed [table 2/figure 2E].\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study assessed the presence of unexplained hypercoagulability in patients well beyond the acute phase of a CVST. We analysed thrombin generation by CAT based on a previous finding of elevated TG levels in 3 patients with CVST seen at the outpatient clinic. Two of the original 3 patients were included in the present study, the third patient was on rivaroxaban and could for that reason not be studied.\u003c/p\u003e\n\u003cp\u003eThrombin generation via de CAT method was performed in order to assess hypercoagulability. The first outcome parameter we considered was lag time. As expected, lag time did not significantly differ between patients and controls, as idiopathic hypercoagulability is mainly reflected by an increase in ETP and peak height (\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eSubsequently we determined ETP, ETP reduction and peak height. ETP did not significantly differ between patients and controls. We tested ETP reduction by adding TM. Binding of thrombin to TM activates Protein C, a potent anticoagulant factor (\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e). We did not observe a significant difference in ETP reduction.\u003c/p\u003e\n\u003cp\u003eIn addition, peak height of the thrombin generation curve was determined. Although there is a visual difference between the two groups (Fig.\u0026nbsp;2B), this did not reach statistical significance, probably due to the small sample size.\u003c/p\u003e\n\u003cp\u003eBased on a previous study which showed increased ETP and peak height in patients after deep venous thrombosis up till two years after the event (\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e), we expected to detect a similar difference in our study population. Other studies do support the findings of increased ETP and peak height after a thrombotic event (\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e). The clinical relevance would be found in the ability to predict recurrent thrombotic events. However, results regarding the predictive value of the thrombin generation assay on development of a secondary event (venous thrombotic event) vary widely (\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e). It should be mentioned that some studies used whole blood, others used PPP or platelet rich plasma, which makes it difficult to compare the outcomes. Nevertheless, we can conclude that the role of thrombin generation as a predictor is still uncertain.\u003c/p\u003e\n\u003cp\u003ePatients with a first episode of CVST, a potentially devastating condition, are usually treated with anticoagulation for a limited time period, ranging from 3 to 6 months. This time is defined, considering the risk of recurrent disease after cessation of anticoagulation as low. However, this risk is not negligible and incidences ranging from 3 to 18 percent \u0026ndash; depending on the risk factors \u0026ndash; for development of recurrence CVST and/or other venous thrombotic events (VTE) have been reported (\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e). Literature suggests that the risk of recurrence is highest up till one till two year(s) after the event (\u003cspan class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e). Therefore, characterizing thrombosis risk after stopping anticoagulation may be useful.\u003c/p\u003e\n\u003cp\u003eIn all patients with CVST, avoidable risk factors like oral contraceptives are typically recorded and eliminated, whereas in younger individual\u0026rsquo;s variable thrombophilia screening is done. Since thrombin generation might have been a convenient single test for detecting thrombophilia, we focused on its use in this population.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy limitations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLimitations of this study include the small number of participants remaining after an extensive search and selection process. CVST is a relatively rare disease and we selected an even smaller group by only including idiopathic CVST patients. The direction of some of the TG comparisons showed a trend towards hypercoagulability in the patients and it may be assumed that in a larger population true differences may emerge. Furthermore, we excluded patients with a severe course of the disease, as we excluded disabled/incapacitated subjects. This might have led to selection bias in the current population. Finally, the time after the event might be too long to be able to detect hypercoagulability and in case of risk factors like oral contraceptives, these were eliminated after the first event. We included patients after three years on average. It might simply be that the hypercoagulable state tends to normalize after this time interval.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this pilot study we did not find any significant aberration in haemostasis between patients that suffered from CVST in the past, and age/gender machted controls. This is potentially due to a small sample size and relatively long follow-up time. Given the fact that CVST is a serious condition with a more than negligible risk of (VTE) recurrence, exclusion of clinically relevant hypercoagulability remains a challenging topic to further study at the acute and later time points, particularly in patients with idiopathic CVST.\u003c/p\u003e"},{"header":"List Of Abbreviations","content":"\u003cp\u003eCAT Calibrated automated thrombogram\u003c/p\u003e\n\u003cp\u003eCVST Cerebral venous sinus thrombosis\u003c/p\u003e\n\u003cp\u003eDBC Diagnosis-treatment combinations\u003c/p\u003e\n\u003cp\u003eETP Endogenous thrombin potential\u003c/p\u003e\n\u003cp\u003eMUMC Maastricht university medical centre\u003c/p\u003e\n\u003cp\u003ePPP Platelet poor plasma\u003c/p\u003e\n\u003cp\u003eTM Thrombomodulin\u003c/p\u003e\n\u003cp\u003eVTE Venous thrombotic event\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cem\u003eEthics approval and consent to participate\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe study was performed at the MUMC and approved by the local medical ethical committee (Medical ethical committee MUMC, approval number; NL 63775.068.17 All participants gave written informed consent.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAvailability of data and materials\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNo specific funding was obtained for this study.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAuthors\u0026apos; contributions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMB, RO, RJO, and HT conceived and designed the study. MB, BK and RO performed the measurements. MB, RO and HT analysed the data and interpreted the results. MB drafted the work. MB, BK, RO, RJO, and HT critically edited and revised the manuscript. MB, BK, RO, RJO, and HT have approved the final version of the manuscript and agree to be accountable for their contributions.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAcknowledgements\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eWeimar C. Diagnosis and treatment of cerebral venous and sinus thrombosis. Curr Neurol Neurosci Rep. 2014;14(1):417.\u003c/li\u003e\n \u003cli\u003eSilvis SM, de Sousa DA, Ferro JM, Coutinho JM. Cerebral venous thrombosis. Nat Rev Neurol. 2017;13(9):555-65.\u003c/li\u003e\n \u003cli\u003eGao L, Xu W, Li T, Yu X, Cao S, Xu H, et al. Accuracy of magnetic resonance venography in diagnosing cerebral venous sinus thrombosis. Thromb Res. 2018;167:64-73.\u003c/li\u003e\n \u003cli\u003eFerro JM, Canhao P, Stam J, Bousser MG, Barinagarrementeria F, Investigators I. Prognosis of cerebral vein and dural sinus thrombosis: results of the International Study on Cerebral Vein and Dural Sinus Thrombosis (ISCVT). Stroke. 2004;35(3):664-70.\u003c/li\u003e\n \u003cli\u003eBreteau G, Mounier-Vehier F, Godefroy O, Gauvrit JY, Mackowiak-Cordoliani MA, Girot M, et al. Cerebral venous thrombosis 3-year clinical outcome in 55 consecutive patients. J Neurol. 2003;250(1):29-35.\u003c/li\u003e\n \u003cli\u003eGameiro J, Ferro JM, Canhao P, Stam J, Barinagarrementeria F, Lindgren A, et al. Prognosis of cerebral vein thrombosis presenting as isolated headache: early vs. late diagnosis. Cephalalgia. 2012;32(5):407-12.\u003c/li\u003e\n \u003cli\u003eFranchini M, Liumbruno GM, Pezzo M. COVID-19 vaccine-associated immune thrombosis and thrombocytopenia (VITT): Diagnostic and therapeutic recommendations for a new syndrome. Eur J Haematol.n/a(n/a).\u003c/li\u003e\n \u003cli\u003eDentali F, Crowther M, Ageno W. Thrombophilic abnormalities, oral contraceptives, and risk of cerebral vein thrombosis: a meta-analysis. Blood. 2006;107(7):2766-73.\u003c/li\u003e\n \u003cli\u003eLuo Y, Tian, X., \u0026amp; Wang, X. . Diagnosis and Treatment of Cerebral Venous Sinus Thrombosis: A Review. Front Aging Neurosci. 2018;10:2.\u003c/li\u003e\n \u003cli\u003eCoutinho JM, Stam J. How to treat cerebral venous and sinus thrombosis. J Thromb Haemost. 2010;8(5):877-83.\u003c/li\u003e\n \u003cli\u003ePalazzo P, Agius P, Ingrand P, Ciron J, Lamy M, Berthomet A, et al. Venous Thrombotic Recurrence After Cerebral Venous Thrombosis: A Long-Term Follow-Up Study. Stroke. 2017;48(2):321-6.\u003c/li\u003e\n \u003cli\u003eDielis AWJH CE, Spronk HMH, van Oerle R, Hamulya\u0026apos;k K, ten Cate , Rosing J. Coagulation factors and the protein C system as determinants of thrombin generation in a normal population. Thromb Haemost. 2008;6(1):125-31.\u003c/li\u003e\n \u003cli\u003eDuarte RCF, Ferreira CN, Rios DRA, Reis HJD, Carvalho MDG. Thrombin generation assays for global evaluation of the hemostatic system: perspectives and limitations. Rev Bras Hematol Hemoter. 2017;39(3):259-65.\u003c/li\u003e\n \u003cli\u003eHemker HC; Giesen P AR, Regnault V, de Smed E, Lecompte T, B\u0026eacute;guin S. The Calibrated Automated Thrombogram (CAT) A universal routine test for hyper- and hypocoagulability. Pathophysiol Haemos Thromb. 2002;32(5-6):249-53.\u003c/li\u003e\n \u003cli\u003eBloemen S, Zwaveling S, Ten Cate H, Ten Cate-Hoek A, de Laat B. Prediction of bleeding risk in patients taking vitamin K antagonists using thrombin generation testing. PLoS One. 2017;12(5):e0176967.\u003c/li\u003e\n \u003cli\u003eHemker HC, Al Dieri R, Beguin S. Thrombin generation assays: accruing clinical relevance. Curr Opin Hematol. 2004;11(3):170-5.\u003c/li\u003e\n \u003cli\u003eTen Cate H. Thrombin generation in clinical conditions. Thromb Res. 2012;129(3):367-70.\u003c/li\u003e\n \u003cli\u003eLoeffen R, Kleinegris MC, Loubele ST, Pluijmen PH, Fens D, van Oerle R, et al. Preanalytic variables of thrombin generation: towards a standard procedure and validation of the method. J Thromb Haemost. 2012;10(12):2544-54.\u003c/li\u003e\n \u003cli\u003eSpronk HM, Dielis AW, De Smedt E, van Oerle R, Fens D, Prins MH, et al. Assessment of thrombin generation II: Validation of the Calibrated Automated Thrombogram in platelet-poor plasma in a clinical laboratory. Thromb Haemost. 2008;100(2):362-4.\u003c/li\u003e\n \u003cli\u003eSpronk HM, Dielis AW, De Smedt E, van Oerle R, Fens D, Prins MH, et al. Assessment of thrombin generation II: Validation of the Calibrated Automated Thrombogram in platelet-poor plasma in a clinical laboratory. Thromb Haemost. 2008;100(2):362-4.\u003c/li\u003e\n \u003cli\u003eKremers BMM, Birocchi S, van Oerle R, Zeerleder S, Spronk HMH, Mees BME, et al. Searching for a Common Thrombo-Inflammatory Basis in Patients With Deep Vein Thrombosis or Peripheral Artery Disease. Front cardiovasc med. 2019;6(33).\u003c/li\u003e\n \u003cli\u003eButenas S, van\u0026apos;t Veer C, Mann KG. \u0026quot;Normal\u0026quot; thrombin generation. Blood. 1999;94(7):2169-78.\u003c/li\u003e\n \u003cli\u003eHemker HC, Beguin S. Thrombin generation in plasma: its assessment via the endogenous thrombin potential. Thromb Haemost. 1995;74(1):134-8.\u003c/li\u003e\n \u003cli\u003eten Cate-Hoek AJ, Dielis AW, Spronk HM, van Oerle R, Hamulyak K, Prins MH, et al. Thrombin generation in patients after acute deep-vein thrombosis. Thromb Haemost. 2008;100(2):240-5.\u003c/li\u003e\n \u003cli\u003eLutsey PL, Folsom AR, Heckbert SR, Cushman M. Peak thrombin generation and subsequent venous thromboembolism: the Longitudinal Investigation of Thromboembolism Etiology (LITE) study. Thromb Haemost. 2009;7(10):1639-48.\u003c/li\u003e\n \u003cli\u003eMachlus KR, Colby EA, Wu JR, Koch GG, Key NS, Wolberg AS. Effects of tissue factor, thrombomodulin and elevated clotting factor levels on thrombin generation in the calibrated automated thrombogram. Thromb Haemost. 2009;102(5):936-44.\u003c/li\u003e\n \u003cli\u003eJoly BS, Sudrie-Arnaud B, Barbay V, Borg JY, Le Cam Duchez V. Thrombin generation test as a marker for high risk venous thrombosis pregnancies. J Thromb Thrombolysis. 2018;45(1):114-21.\u003c/li\u003e\n \u003cli\u003ePark MS, Spears GM, Bailey KR, Xue A, Ferrara MJ, Headlee A, et al. Thrombin generation profiles as predictors of symptomatic venous thromboembolism after trauma: A prospective cohort study. J Trauma Acute Care Surg. 2017;83(3):381-7.\u003c/li\u003e\n \u003cli\u003eRiva N, Vella K, Hickey K, Bertu L, Zammit D, Spiteri S, et al. Biomarkers for the diagnosis of venous thromboembolism: D-dimer, thrombin generation, procoagulant phospholipid and soluble P-selectin. J Clin Pathol. 2018;71(11):1015-22.\u003c/li\u003e\n \u003cli\u003eVoils SA, Lemon SJ, Jordan J, Riley P, Frye R. Early thrombin formation capacity in trauma patients and association with venous thromboembolism. Thromb Res. 2016;147:13-5.\u003c/li\u003e\n \u003cli\u003eWexels F, Dahl OE, Pripp AH, Seljeflot I. Thrombin Generation in Patients With Suspected Venous Thromboembolism. Clin Appl Thromb Hemost. 2017;23(5):416-21.\u003c/li\u003e\n \u003cli\u003eLippi G, Danese E, Favaloro EJ, Montagnana M, Franchini M. Diagnostics in venous thromboembolism: from origin to future prospects. Semin Thromb Hemost. 2015;41(4):374-81.\u003c/li\u003e\n \u003cli\u003eMiranda B, Ferro JM, Canhao P, Stam J, Bousser MG, Barinagarrementeria F, et al. Venous thromboembolic events after cerebral vein thrombosis. Stroke. 2010;41(9):1901-6.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"thrombosis-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"thrj","sideBox":"Learn more about [Thrombosis Journal](http://thrombosisjournal.biomedcentral.com/)","snPcode":"12959","submissionUrl":"https://submission.nature.com/new-submission/12959/3","title":"Thrombosis Journal","twitterHandle":"@Thrombosis_J","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Cerebral venous sinus thrombosis (CVST), Calibrated Automated Thrombogram, patients, Potential value, exploratory study","lastPublishedDoi":"10.21203/rs.3.rs-757602/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-757602/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Cerebral venous sinus thrombosis (CVST) is a relatively rare, but potentially lethal condition. In approximately 15% of the patients, the cause of CVST remains unclear. Conventional clotting tests such as prothrombin time and activated partial thromboplastin time are not sensitive enough to detect prothrombotic conditions nor mild haemostatic abnormalities. The calibrated automated thrombogram (CAT) is a physiological function test that might be able to detect minor aberrations in haemostasis. Therefore, we aimed to detect the presence of a prothrombotic state in patients who endured idiopathic CVST with the CAT assay. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Adult patients with an idiopathic, radiologically proven CVST that had been admitted during the past 3 years were included in this study. The control group consisted of age/gender matched healthy volunteers. Exclusion criteria were known haematological disorders, malignancy (current/past) or hormonal and anticoagulant therapy recipients. We obtained venous blood samples from all participants following cessation of anticoagulation. Using the CAT assay, we determined lag time, normalized endogenous thrombin potential (ETP), ETP reduction and normalized peak height. In addition, prothrombin concentrations were determined. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e We found no significant differences in lag time (4.7 min [4.5-4.9] vs 5.3 min [3.7-5.7], \u003cem\u003ep = \u003c/em\u003e0.691), normalized ETP (142% [124-148] vs 124% [88-138], \u003cem\u003ep\u003c/em\u003e = 0.222), ETP reduction (29% [26-35] vs 28% [24-58], \u003cem\u003ep \u003c/em\u003e\u0026gt;0.999), and normalized peak height (155% [153-175] vs 137 [94-154], \u003cem\u003ep \u003c/em\u003e= 0.056) between patients and their age/gender matched controls. In addition, prothrombin concentrations did not significantly differ between patients and controls (120% [105-132] vs 127% [87-139], \u003cem\u003ep\u003c/em\u003e\u0026gt;0.999.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e Reasons for absent overt hypercoagulability within this study population may be the small patient sample, long time since the event (e.g. 3 years) and avoidance of acquired risk factors like oral contraception. Given the fact that CVST is a serious condition with a more than negligible risk of venous thrombosis event recurrence, exclusion of clinically relevant hypercoagulability remains a challenging topic to further study at the acute and later time points, particularly in patients with idiopathic CVST.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"Potential Value of the Calibrated Automated Thrombogram in Patients After a Cerebral Venous Sinus Thrombosis; an Exploratory Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-08-03 21:12:07","doi":"10.21203/rs.3.rs-757602/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2021-08-24T00:00:00+00:00","index":1,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"decision","content":"Minor revision","date":"2021-08-24T00:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-08-15T00:00:00+00:00","index":2,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"reviewerAgreed","content":"","date":"2021-08-11T00:00:00+00:00","index":2,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-08-10T20:18:28+00:00","index":0,"fulltext":""},{"type":"reviewerAgreed","content":"","date":"2021-08-10T00:00:00+00:00","index":1,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-08-01T12:03:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-07-30T07:29:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2021-07-27T23:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2021-07-27T23:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Thrombosis Journal","date":"2021-07-27T08:58:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"thrombosis-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"thrj","sideBox":"Learn more about [Thrombosis Journal](http://thrombosisjournal.biomedcentral.com/)","snPcode":"12959","submissionUrl":"https://submission.nature.com/new-submission/12959/3","title":"Thrombosis Journal","twitterHandle":"@Thrombosis_J","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b33777b6-b71b-4981-83a9-f05b3105fdd8","owner":[],"postedDate":"August 3rd, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":6189796,"name":"General Biochemistry"},{"id":6189797,"name":"Internal Medicine"}],"tags":[{"value":"VITT","date":"2021-08-01 21:41:57"}],"updatedAt":"2021-10-22T02:00:16+00:00","versionOfRecord":[],"versionCreatedAt":"2021-08-03 21:12:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-757602","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-757602","identity":"rs-757602","version":["v1"]},"buildId":"omnImTCwR2MFx8CMYfrG7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}