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Dysfunctional fibrinolysis and cerebral venous thrombosis
Accepted Manuscript Dysfunctional fibrinolysis and cerebral venous thrombosis
Aniket Prabhudesai, Shrimati Shetty, Kanjaksha Ghosh, Bipin Kulkarni PII: DOI: Reference:
S1079-9796(17)30058-X doi: 10.1016/j.bcmd.2017.05.007 YBCMD 2193
To appear in:
Blood Cells, Molecules and Diseases
Received date: Revised date: Accepted date:
2 February 2017 11 May 2017 11 May 2017
Please cite this article as: Aniket Prabhudesai, Shrimati Shetty, Kanjaksha Ghosh, Bipin Kulkarni , Dysfunctional fibrinolysis and cerebral venous thrombosis, Blood Cells, Molecules and Diseases (2017), doi: 10.1016/j.bcmd.2017.05.007
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ACCEPTED MANUSCRIPT Full Length Article
Dysfunctional fibrinolysis and cerebral venous thrombosis Aniket Prabhudesai1, Shrimati Shetty1, Kanjaksha Ghosh2, Bipin Kulkarni1
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1 National Institute of Immunohaematology (ICMR), Department of Thrombosis and
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Haemostasis, KEM Hospital, Parel, Mumbai, India.
2 Surat Raktadan Kendra & Research Centre, Regional Blood Transfusion Centre, Surat,
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Gujarat, India.
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Running head: Fibrinolysis and cerebral venous thrombosis
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Corresponding author: Dr. Bipin Kulkarni, PhD, Department of Haemostasis and Thrombosis, National Institute of Immunohaematology (ICMR), 13th floor, New MS Building, KEM Hospital
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campus, Parel, Mumbai, 400 012, India.
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Telephone: +912224138518Fax: +9122 24138521
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Email:[email protected] Tel: +91 22 24138518. Fax- +91 22 24138521
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ACCEPTED MANUSCRIPT Abstract Cerebral venous thrombosis (CVT) is an uncommon neurological disease with high morbidity and mortality. Even after extensive thrombophilia screening, majority of the thrombosis cases remain with unknown etiology. Hypofibrinolysis due to acquired or congenital deficiencies or
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abnormalities in factors in the fibrinolytic cascade is a known cause of thrombosis at any site. In
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the present study 104 cases of radiologically confirmed CVT cases were investigated for the
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conventional thrombophilia along with factors in the fibrinolytic cascade to find a possible etiology for the clinical manifestation. Conventional thrombophilia markers which included PC,
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PS, AT and FVL mutation were detected in 16.3 % of the patients. Approximately 19 % cases
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had grossly elevated plasma PAI-1 levels. PAI-1 4G/4G genotype was found to be strongly associated with high PAI-1 levels. 2.9% cases had reduced tPA levels, 1.9% had plasminogen
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deficiency and 1.9% cases had increased alpha-2-antiplasmin levels. Along with conventional
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thrombophilia, dysfunctional fibrinolysis is found to be strongly associated with CVT. Understanding the role of risk factors is important for appropriate treatment of this serious
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disorder.
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Key words: Cerebral venous thrombosis; Fibrinolysis; PAI-1; Thrombophilia; PAI4G/4G.
Abbreviations:
CVT: cerebral venous thrombosis; PAI-1: plasminogen activator inhibitor -1; PC: protein C; PS: protein S; AT: antithrombin III; FVL: Factor V Leiden; tPA: tissue plasminogen activator; PLG: plasminogen; A2-AP: alpha-2-antiplasmin; APLA: antiphospholipid antibodies; LA: lupus anticoagulant.
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ACCEPTED MANUSCRIPT Introduction Cerebral venous thrombosis (CVT) is a rare cerebrovascular disease which is multifactorial in nature with an annual incidence of 1.32 cases per 100,000 person-years [1]. CVT is difficult to diagnose due to varied clinical presentation and etiologically heterogeneous factors [2]. The
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onset of CVT may be acute, sub-acute or chronic. Mortality rate has been brought down due to
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immediate initiation of anticoagulation therapy in diagnosed CVT patients. Presence of multiple
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risk factors including hereditary thrombophilia markers i.e. protein C (PC), protein S (PS), antithrombin III (AT), factor V Leiden (FVL) mutation, prothrombin G20210A mutation and
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acquired or transient risk factors such as pregnancy, puerperium, antiphospholipid syndrome,
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trauma, surgery, cancer, exogenous hormones, oral contraceptive use, inflammation, infection and hyperhomocysteinemia is known to account for CVT [3-5]. However, these multiple risk
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strong family history or recurrence.
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factors account for a fraction of these cases and many cases still remain unexplained despite of a
Fibrinolysis plays an important role in dissolution of blood clots formed in response to tissue
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injury. Fibrinolytic system consists of activators and inhibitors of plasminogen and their
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molecular interplay regulates fibrinolysis. Dysfunctional fibrinolysis or hypofibrinolysis is a state wherein there is decreased clearance of blood clots thereby leading to a state of thrombosis.
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A clear association has been established between hypofibrinolysis and risk of venous thrombosis in the Leiden Thrombophilia Study [6].
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ACCEPTED MANUSCRIPT Materials and methods One hundred and four patients with a proven diagnosis of CVT based on magnetic resonance imaging (MRI) combined with magnetic resonance venography (MRV), and/or computed tomography (CT) venography from a tertiary care centre in Mumbai were included in the present
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study. This is a prospective study which included (a) patients in acute/ sub acute/ chronic phase
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of CVT who were not started on anticoagulants and (b) patients who had stopped anticoagulation after completing at least three months of anticoagulation therapy. Blood samples obtained from
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patients who had received anticoagulation ranged from 4 months to 13 years relative to CVT (average: 1 year 11 months). The study was approved by the Institutional Ethics Committee
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Review Board in accordance with declaration of Helsinki. A written informed consent was
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obtained from all the participants. Patients were initially screened for thrombophilia markers PC, PS, AT and FVL mutation using standard procedures. Normal range for PC, PS, and AT was
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70–140% and all patients were diagnosed as PC, PS, or AT deficiency if the values were below
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60% [Diagnostica stago, Asnires, France]. Fibrinolysis markers were studied using commercial ELISA kits. [Plasminogen (PLG), Boster biological, CA, USA; tissue plasminogen activator
alpha-2-antiplasmin (A2-AP), Bioassay technologies, Shanghai, China]. Normal
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MI, USA;
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(tPA), Assaypro, MO, USA; plasminogen activator inhibitor-1 (PAI-1), Molecular Innovations,
ranges were set for fibrinolytic parameters on studying 50 healthy controls. Patients were diagnosed with high PAI-1 if values were above 110 ng/ml, with reduced tPA if values were below 0.75 ng/ml, with plasminogen deficiency if values were below 300µg/ml and with high A2-AP if values were above 200 ng/ml. Homocysteine levels were measured by chemiluminescence immunoassay [Normal range: 3.7-13.9 µmol/L, above which was considered hyperhomocysteinemia]. Lupus anticoagulant screening was done using LA1 and LA2 reagents 4
ACCEPTED MANUSCRIPT (Siemens Healthcare GmbH, Erlangen, Germany). FVL mutation [rs6025] was studied using PCR-RFLP method (MnlI digestion) and PAI-1 4G/5G promoter polymorphism [rs1799768] was
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studied using allele specific PCR.
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ACCEPTED MANUSCRIPT Results Mean patient age in the study was 32.7 ± 11.8 years. Study included sixty four (61.5%) male patients and forty (38.5%) female patients with a mean age of 34.3 ± 11.7 and 30.9 ± 11.9 years respectively. Thrombosis was predominantly seen in superior sagittal sinus (60.4%), followed by
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transverse sinus (57.3%), cortical veins (24%), extending to internal jugular veins (12.5%),
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straight sinus (10.4%), vein of Galen and internal cerebral veins (8.3%) and cavernous sinus
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(2.1%). The major clinical manifestations were headache (56.4%), convulsions (38.5%), blurring of vision (15.4%), vomiting (12.8%), altered consciousness (10.2%), hemiparesis (7.7%), vertigo
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and migraine (5.1%).
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Hereditary thrombophilia was present in 17 (16.3%) cases.
Seven (6.7%) cases were PC
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deficient; four (3.8%) were PS deficient (all females). AT deficiency was seen in one (1%) case. None of the patients had a combined deficiency of the natural anticoagulants in the present study.
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Five (4.8%) cases had heterozygous FVL mutation. APLA (antiphospholipid antibodies) and LA
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(lupus anticoagulant) were present in 6 cases (5.8%).
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Twenty (19.2%) patients had hyperhomocysteinemia. Fourteen (13.5%) patients had high CRP levels (>10mg/L). Seven (6.7%) patients had hypertension, 17 (16.3%) had trauma (recent
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history of fall/ head injury/ accident/ surgery); 2 (5%) patients had history of pregnancy loss and pre-eclampsia while 2 (5%) were on oral contraceptive pills. Three (2.9%) patients had tuberculosis and two (1.9%) had suffered from typhoid fever. Two patients (1.9%) had polycythemia vera. Of all the fibrinolytic markers studied, 20 (19.2%) patients had grossly elevated levels of PAI-1 levels. High PAI-1 is found to be a risk factor for CVT (P=0.0182, OR=11.6) in the present
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ACCEPTED MANUSCRIPT study. High plasma PAI-1 level has been attributed to the 4G allele of 4G/5G promoter polymorphism [7]. In our study, 19 (18.2%) patients had a homozygous 4G/4G PAI-1 promoter polymorphism. Patients with a 4G/4G genotype had significantly high mean of 117.6 ± 83.6 ng/ml PAI-1 level when compared to those with 4G/5G genotype (mean: 62.5 ± 42.1 ng/ml) and
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5G/5G genotype (mean: 53.3 ± 40.7 ng/ml) (P=0.0103, Kruskal Wallis test, Figure 1). Three
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(2.9%) patients had reduced tPA levels. Two (1.9%) cases had plasminogen deficiency. Two
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(1.9%) cases had increased A2-AP. Demographic and laboratory data of CVT patients of the
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present study along with the scatter plots is shown in Table 1 and Figure 2. Presence of multiple risk factors was seen in five cases (4.8%) [Table 2]. Two patients with a
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history of familial thrombosis were enrolled in the study. Of these, first patient had PS deficiency
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along with homozygous 4G/4G PAI-1 polymorphism and high PAI-1 levels. Second patient had PC deficiency and hyperhomocysteinemia. His elder brother (also diagnosed as PC deficiency)
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had an episode of deep vein thrombosis.
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ACCEPTED MANUSCRIPT Discussion In the present study it was seen that 16.3% CVT cases had a hereditary thrombophilia marker. PC deficiency was the most prevalent thrombophilia marker seen in 6.7% CVT cases. PS and AT deficiency was noted in 3.8% and 1% cases respectively. Prevalence of PC, PS and AT
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deficiencies has been reported to range between 0% - 20% in CVT cases in several earlier
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studies. The prevalence of FVL heterozygous mutation was 4.8% in the present study as against 3% - 17.3% prevalence in CVT cases reported from various countries [Table 3]. Overall
strong
association
between
CVT
and
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prevalence of FVL mutation in Indian population is very low. Dentali F et al. have shown a FVL,
Prothrombin
G20210A
mutation
and
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hyperhomocysteinemia [16]. Prothrombin G20210A mutation is a risk factor associated with
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CVT [17]. The mutation has not been analyzed in the present study, since in earlier studies it has
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been shown that either the mutation is too rare or not present in Indian population [18].
Fibrinolysis is a regulation mechanism which ensures dissolution of blood clots and maintaining
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the vascular system. Functioning of the fibrinolytic system revolves around the central enzyme
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‘plasmin’. Plasminogen, an inactive proenzyme, is acted upon and converted to plasmin in the presence of tPA which in turn degrades fibrin strands into soluble fibrin degradation products.
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Plasminogen activator inhibitor-1 (PAI-1) and thrombin activatable fibrinolysis inhibitor (TAFI) act on plasminogen whereas A2-AP directly inhibits plasmin enzyme. Hypofibrinolysis is the result of impaired activation of the fibrinolytic system which may lead to thrombotic complications [19-21]. However, the role of individual fibrinolytic markers in venous thrombosis has not been established or has been contradicting in literature. In our study, 19.2% cases had grossly elevated PAI-1 levels which could have led to a hypofibrinolytic state resulting in CVT (P=0.0182, OR=11.6). Stegnar et al reported increased PAI-1 in up to 40% VTE cases leading to 8
ACCEPTED MANUSCRIPT impaired fibrinolysis [7]. PAI-1 is an acute phase protein and raised levels of PAI-1 are known to associate with large spectrum of disease states [22]. Therefore, the possibility of raised PAI-1 as secondary to CVT cannot be ruled out. The 4G allele of 4G/5G promoter polymorphism of PAI1 has been linked with increased plasma PAI-1 levels, which may lead to hypofibrinolysis,
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thereby increasing the incidence of thrombotic events [23, 24]. Of the twenty (19.2%) patients
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which showed increased PAI-1 values, eleven (10.5%) were in acute/sub acute/chronic phase of
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CVT when sample was collected. However, seven (6.7%) of those eleven patients also had presence of 4G/4G PAI-1 genotype. So, increased PAI-1 could have been a result of presence of
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4G/4G genotype in those patients. There are contradictory reports on association of tPA and risk
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of thrombosis. Meltzer et al. reported an association between increased tPA levels and venous thrombosis however, the authors also stated that this increase in tPA could be a result of
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inflammation and endothelial activation [25]. Tezzon et al have reported a CVT case with tPA
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deficiency due to defective tPA release [26]. In the present study, we found 2.9% cases to have reduced tPA levels (P=0.74, OR=1.45) and were not significantly different in patients 2.1 ±
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1.5ng/ml and 1.9 ± 1.1ng/ml in controls (P=0.44). The role of plasminogen in thrombosis risk
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has remained unclear due to contradictory reports in the literature. 1.9% cases had plasminogen deficiency (mean: 262.5 ± 17.7 µg/ml) in the present study. Both patients and controls showed
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almost similar levels of plasminogen (mean: 601.1 ± 177 µg/ml in patients versus 604.8 ± 172 µg/ml in controls; P=0.43). Schutta et al had previously reported a CVT patient with reduced plasminogen activity level [27]. High levels of A2-AP were seen in 1.9% cases in this study (mean: 287.1 ± 62.4 ng/ml). Lisman et al had reported that hypofibrinolysis caused by elevated levels of A2-AP may contribute to arterial thrombosis whereas PAI-1 and TAFI might be associated with venous thrombosis risk [28]. Hyperhomocysteinemia has been associated with a
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ACCEPTED MANUSCRIPT 4-fold increased risk of CVT [10]. Variants of MTHFR are known to be associated with high levels of homocysteine. In the present study, 19.2% cases had hyperhomocysteinemia.
Dentali F et al have shown that the risk of CVT in women taking oral contraceptives is 6-fold higher than that of non-users [16]. APLA and LA known to be prothrombotic in clinical
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manifestation were present in 5.8% cases. Hypertension was seen in 6.7% cases of which 2.9%
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cases also had a 4G/4G PAI-1 promoter polymorphism and high PAI-1 levels. Systemic infectious diseases like tuberculosis (2.9%) and typhoid (1.9%) along with polycythemia vera
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(1.9%) and recent trauma (16.3%) were some other acquired causes of CVT in these patients.
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ACCEPTED MANUSCRIPT Conclusion
Cerebral venous thrombosis is a multifactorial disease. Hereditary thrombophilia was seen in 16.3% of the cases. In addition to genetic, non-genetic and transient risk factors, dysfunctional fibrinolysis due to increased PAI-1 levels has been found to be strongly associated with CVT in
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the present study. Hypofibrinolysis with high PAI-1 levels mainly associated with PAI-1 4G/4G
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promoter polymorphism contributed to 19.2% of the CVT cases. Inflammation may also play a role in establishing an acquired state of hypercoagulabitity and hypofibrinolysis. Although
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genetic fibrinolytic disorders are rare, acquired cases of hypofibrinolysis seem to result in clinical manifestation of thrombosis. Combination of both heritable and acquired thrombophilia
fibrinolytic pathway will facilitate in a better
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markers both in the anticoagulant and
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along with hypofibrinolysis have a greater risk of CVT. Investigation of a combination of
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understanding of the pathophysiological mechanisms of cerebral venous thrombosis. Though CVT patients with abnormal levels of fibrinolytic and coagulation proteins were rechecked and
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reconfirmed yet the lack of a follow up study is a limitation of the study.
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ACCEPTED MANUSCRIPT Funding: This work is supported by the regular intramural funding of Indian Council of Medical Research (ICMR).
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Conflict of interest: None of the authors have conflict of interests to be declared.
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Authorship: The manuscript represents valid work. All authors certify that they have
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participated sufficiently in the work to take public responsibility for the content, including participation in the concept, design, analysis, writing, or revision of the manuscript. Furthermore,
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each author certifies that this material or similar material has not been and will not be submitted
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to or published in any other publication.
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Author contribution:
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BK, SS, KG conceived and designed the experiments. AP performed the experiments and analysed the data.
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AP, BK and SS wrote the paper.
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ACCEPTED MANUSCRIPT Figure Legend: Figure 1: Comparison of PAI-1 level with genotypes of PAI-4G/5G promoter polymorphism. There is a significant difference of PAI-1 (Plasminogen activator inhibitor-1) levels observed in
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4G/4G when compared to 4G/5G and 5G/5G genotypes (p=0.0103, Kruskal Wallis test).
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Figure 2: Scatter plots of laboratory values (thrombophilia and fibrinolysis parameters) of CVT
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patients in the present study.
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Table 1: Demographic and laboratory data of CVT cases in the present study 104 32.7 ± 11.8
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CR
7 (6.7%) 4 (3.8%) 1 (1%) 5 (4.8%)
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64 (61.5%) 40 (38.5%)
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20 (19.2%) 19 (18.2%) 3 (2.9%) 2 (1.9%) 2 (1.9%)
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CVT cases Age (years) Sex Male Female Hereditary Thrombophilia PC deficiency (<60%) PS deficiency (<60%) AT deficiency (<60%) FVL mutation Hypofibrinolysis High PAI-1 (>110 ng/ml) PAI-1 4G/4G polymorphism Reduced tPA (<0.75 ng/ml) PLG deficiency (<300 µg/ml) High A2-AP (>200 ng/ml) Acquired or transient risk factors Hyperhomocysteinemia Trauma High CRP (>10 mg/L) Hypertension APLA/ LA Tuberculosis Typhoid Oral contraceptive use Pre-eclampsia History of pregnancy loss Polycythemia vera
20 (19.2%) 17 (16.3%) 14 (13.5%) 7 (6.7%) 6 (5.8%) 3 (2.9%) 2 (1.9%) 2 (5%)* 2 (5%)* 2 (5%)* 2 (1.9%)
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Abbreviations: PC: Protein C; PS- Protein S; AT: Antithrombin III, FVL: Factor V Leiden, PLGPlasminogen, PAI-1: Plasminogen activator inhibitor-1, tPA- Tissue plasminogen activator, A2-AP: Alpha-2-antiplasmin, APLA: Antiphospholipid antibodies, LA: Lupus Anticoagulant, CRP: C-reactive protein. * Risk factors in females alone (out of forty).
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ACCEPTED MANUSCRIPT Table 2: 5 CVT (4.8%) patients with combination of both hereditary and fibrinolysis markers Patient
Age (yr)
Sex
Combined Risk factors
AC
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PT E
D
MA
NU
SC
RI
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1 20 F PS deficiency, Reduced tPA, Oral contraceptive use 2 5 F PC deficiency, High PAI-1, PAI-1 4G/4G, High CRP 3 43 F PS deficiency, High PAI-1, PAI-1 4G/4G,Hyperhomocysteinemia 4 43 M FVL mutation, High PAI-1 5 36 M PC deficiency, High PAI-1, PAI-1 4G/4G and High A2-AP Abbreviation: PC: Protein C; PS- Protein S; FVL: Factor V Leiden, PAI-1: Plasminogen activator inhibitor-1, tPATissue plasminogen activator, A2-AP: Alpha-2-antiplasmin, CRP: C-reactive protein.
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ACCEPTED MANUSCRIPT Table 3: Comparison of the risk factors of the present study with other various studies Reference
[4]
[5]
[8]
[9]
[10]
[11]
[12]
[13]
Country
India
France
Tunisia
Poland
Italy
Turkey
United States
Iran
Number of patients
612
187
160
163
154
75
182
Study Design PC deficiency (%) PS deficiency (%) AT deficiency (%) FVL mutation (%)
R 8.5 4.8 2 3.1
P DN DN DN 8
R 1.8 4.3 1.8 DN
R 0 4 1 10
R 5.2 3.1 2.5 12.4
R 14.7 2 0 17.3
M 2 4 1 3
APLA/LA (%)
DN DN
11 DN
DN 7.5
11 1.8
21.5 4
Hyperhomocysteinemia (%)
DN
28
Infection (%)
6.5
16
DN 8.7
Trauma/ surgery (%)
-
5 5 10 57
1.8 3.7 31.8 23.5
PT gene G20210A (%)
4 14 12
D E
27 2
M
A
5
U N
C S
R 2.4 1.6 DN DN
13.3 DN
DN DN
DN 3.2
32 4
5 2
0.8 8.9
1
Pakistan
Germany
India
109
149
104
R 6 8 7 DN
P 4 5.4 3.4 14.8
P 6.7 3.8 1 4.8
DN 4
4.7 DN
DN 5.8
DN 29.5
19.2 1.9
29 18
9.67 9.4 16.3 Cancer (%) 2 10 7 Hematological causes (%) 1.6 1.9 OC use (%)* DN 34 71 5 46 3 5 Pregnancy/Postpartum/ adverse pregnancy 8 3 38.7 10 33.9 7 8.1 17 10 conditions(%)* Abbreviation: PC: Protein C, PS- Protein S, AT: Antithrombin III, FVL: Factor V Leiden, PT:Prothrombin, APLA: Antiphospholipid antibodies, LA: Lupus Anticoagulant, ACLA: Anticardiolipin antibodies, OC: Oral contraceptive, DN: Data not available , R- Retrospective, P- Prospective, M- Mixed * Risk factors in females alone
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Aniket Prabhudesai, Shrimati Shetty, Kanjaksha Ghosh, Bipin Kulkarni PII: DOI: Reference:
S1079-9796(17)30058-X doi: 10.1016/j.bcmd.2017.05.007 YBCMD 2193
To appear in:
Blood Cells, Molecules and Diseases
Received date: Revised date: Accepted date:
2 February 2017 11 May 2017 11 May 2017
Please cite this article as: Aniket Prabhudesai, Shrimati Shetty, Kanjaksha Ghosh, Bipin Kulkarni , Dysfunctional fibrinolysis and cerebral venous thrombosis, Blood Cells, Molecules and Diseases (2017), doi: 10.1016/j.bcmd.2017.05.007
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ACCEPTED MANUSCRIPT Full Length Article
Dysfunctional fibrinolysis and cerebral venous thrombosis Aniket Prabhudesai1, Shrimati Shetty1, Kanjaksha Ghosh2, Bipin Kulkarni1
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1 National Institute of Immunohaematology (ICMR), Department of Thrombosis and
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Haemostasis, KEM Hospital, Parel, Mumbai, India.
2 Surat Raktadan Kendra & Research Centre, Regional Blood Transfusion Centre, Surat,
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Gujarat, India.
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Running head: Fibrinolysis and cerebral venous thrombosis
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Corresponding author: Dr. Bipin Kulkarni, PhD, Department of Haemostasis and Thrombosis, National Institute of Immunohaematology (ICMR), 13th floor, New MS Building, KEM Hospital
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campus, Parel, Mumbai, 400 012, India.
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Telephone: +912224138518Fax: +9122 24138521
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Email:[email protected] Tel: +91 22 24138518. Fax- +91 22 24138521
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ACCEPTED MANUSCRIPT Abstract Cerebral venous thrombosis (CVT) is an uncommon neurological disease with high morbidity and mortality. Even after extensive thrombophilia screening, majority of the thrombosis cases remain with unknown etiology. Hypofibrinolysis due to acquired or congenital deficiencies or
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abnormalities in factors in the fibrinolytic cascade is a known cause of thrombosis at any site. In
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the present study 104 cases of radiologically confirmed CVT cases were investigated for the
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conventional thrombophilia along with factors in the fibrinolytic cascade to find a possible etiology for the clinical manifestation. Conventional thrombophilia markers which included PC,
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PS, AT and FVL mutation were detected in 16.3 % of the patients. Approximately 19 % cases
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had grossly elevated plasma PAI-1 levels. PAI-1 4G/4G genotype was found to be strongly associated with high PAI-1 levels. 2.9% cases had reduced tPA levels, 1.9% had plasminogen
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deficiency and 1.9% cases had increased alpha-2-antiplasmin levels. Along with conventional
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thrombophilia, dysfunctional fibrinolysis is found to be strongly associated with CVT. Understanding the role of risk factors is important for appropriate treatment of this serious
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disorder.
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Key words: Cerebral venous thrombosis; Fibrinolysis; PAI-1; Thrombophilia; PAI4G/4G.
Abbreviations:
CVT: cerebral venous thrombosis; PAI-1: plasminogen activator inhibitor -1; PC: protein C; PS: protein S; AT: antithrombin III; FVL: Factor V Leiden; tPA: tissue plasminogen activator; PLG: plasminogen; A2-AP: alpha-2-antiplasmin; APLA: antiphospholipid antibodies; LA: lupus anticoagulant.
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ACCEPTED MANUSCRIPT Introduction Cerebral venous thrombosis (CVT) is a rare cerebrovascular disease which is multifactorial in nature with an annual incidence of 1.32 cases per 100,000 person-years [1]. CVT is difficult to diagnose due to varied clinical presentation and etiologically heterogeneous factors [2]. The
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onset of CVT may be acute, sub-acute or chronic. Mortality rate has been brought down due to
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immediate initiation of anticoagulation therapy in diagnosed CVT patients. Presence of multiple
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risk factors including hereditary thrombophilia markers i.e. protein C (PC), protein S (PS), antithrombin III (AT), factor V Leiden (FVL) mutation, prothrombin G20210A mutation and
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acquired or transient risk factors such as pregnancy, puerperium, antiphospholipid syndrome,
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trauma, surgery, cancer, exogenous hormones, oral contraceptive use, inflammation, infection and hyperhomocysteinemia is known to account for CVT [3-5]. However, these multiple risk
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strong family history or recurrence.
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factors account for a fraction of these cases and many cases still remain unexplained despite of a
Fibrinolysis plays an important role in dissolution of blood clots formed in response to tissue
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injury. Fibrinolytic system consists of activators and inhibitors of plasminogen and their
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molecular interplay regulates fibrinolysis. Dysfunctional fibrinolysis or hypofibrinolysis is a state wherein there is decreased clearance of blood clots thereby leading to a state of thrombosis.
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A clear association has been established between hypofibrinolysis and risk of venous thrombosis in the Leiden Thrombophilia Study [6].
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ACCEPTED MANUSCRIPT Materials and methods One hundred and four patients with a proven diagnosis of CVT based on magnetic resonance imaging (MRI) combined with magnetic resonance venography (MRV), and/or computed tomography (CT) venography from a tertiary care centre in Mumbai were included in the present
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study. This is a prospective study which included (a) patients in acute/ sub acute/ chronic phase
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of CVT who were not started on anticoagulants and (b) patients who had stopped anticoagulation after completing at least three months of anticoagulation therapy. Blood samples obtained from
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patients who had received anticoagulation ranged from 4 months to 13 years relative to CVT (average: 1 year 11 months). The study was approved by the Institutional Ethics Committee
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Review Board in accordance with declaration of Helsinki. A written informed consent was
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obtained from all the participants. Patients were initially screened for thrombophilia markers PC, PS, AT and FVL mutation using standard procedures. Normal range for PC, PS, and AT was
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70–140% and all patients were diagnosed as PC, PS, or AT deficiency if the values were below
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60% [Diagnostica stago, Asnires, France]. Fibrinolysis markers were studied using commercial ELISA kits. [Plasminogen (PLG), Boster biological, CA, USA; tissue plasminogen activator
alpha-2-antiplasmin (A2-AP), Bioassay technologies, Shanghai, China]. Normal
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MI, USA;
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(tPA), Assaypro, MO, USA; plasminogen activator inhibitor-1 (PAI-1), Molecular Innovations,
ranges were set for fibrinolytic parameters on studying 50 healthy controls. Patients were diagnosed with high PAI-1 if values were above 110 ng/ml, with reduced tPA if values were below 0.75 ng/ml, with plasminogen deficiency if values were below 300µg/ml and with high A2-AP if values were above 200 ng/ml. Homocysteine levels were measured by chemiluminescence immunoassay [Normal range: 3.7-13.9 µmol/L, above which was considered hyperhomocysteinemia]. Lupus anticoagulant screening was done using LA1 and LA2 reagents 4
ACCEPTED MANUSCRIPT (Siemens Healthcare GmbH, Erlangen, Germany). FVL mutation [rs6025] was studied using PCR-RFLP method (MnlI digestion) and PAI-1 4G/5G promoter polymorphism [rs1799768] was
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studied using allele specific PCR.
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ACCEPTED MANUSCRIPT Results Mean patient age in the study was 32.7 ± 11.8 years. Study included sixty four (61.5%) male patients and forty (38.5%) female patients with a mean age of 34.3 ± 11.7 and 30.9 ± 11.9 years respectively. Thrombosis was predominantly seen in superior sagittal sinus (60.4%), followed by
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transverse sinus (57.3%), cortical veins (24%), extending to internal jugular veins (12.5%),
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straight sinus (10.4%), vein of Galen and internal cerebral veins (8.3%) and cavernous sinus
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(2.1%). The major clinical manifestations were headache (56.4%), convulsions (38.5%), blurring of vision (15.4%), vomiting (12.8%), altered consciousness (10.2%), hemiparesis (7.7%), vertigo
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and migraine (5.1%).
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Hereditary thrombophilia was present in 17 (16.3%) cases.
Seven (6.7%) cases were PC
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deficient; four (3.8%) were PS deficient (all females). AT deficiency was seen in one (1%) case. None of the patients had a combined deficiency of the natural anticoagulants in the present study.
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Five (4.8%) cases had heterozygous FVL mutation. APLA (antiphospholipid antibodies) and LA
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(lupus anticoagulant) were present in 6 cases (5.8%).
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Twenty (19.2%) patients had hyperhomocysteinemia. Fourteen (13.5%) patients had high CRP levels (>10mg/L). Seven (6.7%) patients had hypertension, 17 (16.3%) had trauma (recent
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history of fall/ head injury/ accident/ surgery); 2 (5%) patients had history of pregnancy loss and pre-eclampsia while 2 (5%) were on oral contraceptive pills. Three (2.9%) patients had tuberculosis and two (1.9%) had suffered from typhoid fever. Two patients (1.9%) had polycythemia vera. Of all the fibrinolytic markers studied, 20 (19.2%) patients had grossly elevated levels of PAI-1 levels. High PAI-1 is found to be a risk factor for CVT (P=0.0182, OR=11.6) in the present
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ACCEPTED MANUSCRIPT study. High plasma PAI-1 level has been attributed to the 4G allele of 4G/5G promoter polymorphism [7]. In our study, 19 (18.2%) patients had a homozygous 4G/4G PAI-1 promoter polymorphism. Patients with a 4G/4G genotype had significantly high mean of 117.6 ± 83.6 ng/ml PAI-1 level when compared to those with 4G/5G genotype (mean: 62.5 ± 42.1 ng/ml) and
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5G/5G genotype (mean: 53.3 ± 40.7 ng/ml) (P=0.0103, Kruskal Wallis test, Figure 1). Three
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(2.9%) patients had reduced tPA levels. Two (1.9%) cases had plasminogen deficiency. Two
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(1.9%) cases had increased A2-AP. Demographic and laboratory data of CVT patients of the
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present study along with the scatter plots is shown in Table 1 and Figure 2. Presence of multiple risk factors was seen in five cases (4.8%) [Table 2]. Two patients with a
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history of familial thrombosis were enrolled in the study. Of these, first patient had PS deficiency
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along with homozygous 4G/4G PAI-1 polymorphism and high PAI-1 levels. Second patient had PC deficiency and hyperhomocysteinemia. His elder brother (also diagnosed as PC deficiency)
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had an episode of deep vein thrombosis.
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ACCEPTED MANUSCRIPT Discussion In the present study it was seen that 16.3% CVT cases had a hereditary thrombophilia marker. PC deficiency was the most prevalent thrombophilia marker seen in 6.7% CVT cases. PS and AT deficiency was noted in 3.8% and 1% cases respectively. Prevalence of PC, PS and AT
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deficiencies has been reported to range between 0% - 20% in CVT cases in several earlier
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studies. The prevalence of FVL heterozygous mutation was 4.8% in the present study as against 3% - 17.3% prevalence in CVT cases reported from various countries [Table 3]. Overall
strong
association
between
CVT
and
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prevalence of FVL mutation in Indian population is very low. Dentali F et al. have shown a FVL,
Prothrombin
G20210A
mutation
and
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hyperhomocysteinemia [16]. Prothrombin G20210A mutation is a risk factor associated with
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CVT [17]. The mutation has not been analyzed in the present study, since in earlier studies it has
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been shown that either the mutation is too rare or not present in Indian population [18].
Fibrinolysis is a regulation mechanism which ensures dissolution of blood clots and maintaining
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the vascular system. Functioning of the fibrinolytic system revolves around the central enzyme
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‘plasmin’. Plasminogen, an inactive proenzyme, is acted upon and converted to plasmin in the presence of tPA which in turn degrades fibrin strands into soluble fibrin degradation products.
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Plasminogen activator inhibitor-1 (PAI-1) and thrombin activatable fibrinolysis inhibitor (TAFI) act on plasminogen whereas A2-AP directly inhibits plasmin enzyme. Hypofibrinolysis is the result of impaired activation of the fibrinolytic system which may lead to thrombotic complications [19-21]. However, the role of individual fibrinolytic markers in venous thrombosis has not been established or has been contradicting in literature. In our study, 19.2% cases had grossly elevated PAI-1 levels which could have led to a hypofibrinolytic state resulting in CVT (P=0.0182, OR=11.6). Stegnar et al reported increased PAI-1 in up to 40% VTE cases leading to 8
ACCEPTED MANUSCRIPT impaired fibrinolysis [7]. PAI-1 is an acute phase protein and raised levels of PAI-1 are known to associate with large spectrum of disease states [22]. Therefore, the possibility of raised PAI-1 as secondary to CVT cannot be ruled out. The 4G allele of 4G/5G promoter polymorphism of PAI1 has been linked with increased plasma PAI-1 levels, which may lead to hypofibrinolysis,
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thereby increasing the incidence of thrombotic events [23, 24]. Of the twenty (19.2%) patients
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which showed increased PAI-1 values, eleven (10.5%) were in acute/sub acute/chronic phase of
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CVT when sample was collected. However, seven (6.7%) of those eleven patients also had presence of 4G/4G PAI-1 genotype. So, increased PAI-1 could have been a result of presence of
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4G/4G genotype in those patients. There are contradictory reports on association of tPA and risk
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of thrombosis. Meltzer et al. reported an association between increased tPA levels and venous thrombosis however, the authors also stated that this increase in tPA could be a result of
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inflammation and endothelial activation [25]. Tezzon et al have reported a CVT case with tPA
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deficiency due to defective tPA release [26]. In the present study, we found 2.9% cases to have reduced tPA levels (P=0.74, OR=1.45) and were not significantly different in patients 2.1 ±
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1.5ng/ml and 1.9 ± 1.1ng/ml in controls (P=0.44). The role of plasminogen in thrombosis risk
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has remained unclear due to contradictory reports in the literature. 1.9% cases had plasminogen deficiency (mean: 262.5 ± 17.7 µg/ml) in the present study. Both patients and controls showed
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almost similar levels of plasminogen (mean: 601.1 ± 177 µg/ml in patients versus 604.8 ± 172 µg/ml in controls; P=0.43). Schutta et al had previously reported a CVT patient with reduced plasminogen activity level [27]. High levels of A2-AP were seen in 1.9% cases in this study (mean: 287.1 ± 62.4 ng/ml). Lisman et al had reported that hypofibrinolysis caused by elevated levels of A2-AP may contribute to arterial thrombosis whereas PAI-1 and TAFI might be associated with venous thrombosis risk [28]. Hyperhomocysteinemia has been associated with a
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ACCEPTED MANUSCRIPT 4-fold increased risk of CVT [10]. Variants of MTHFR are known to be associated with high levels of homocysteine. In the present study, 19.2% cases had hyperhomocysteinemia.
Dentali F et al have shown that the risk of CVT in women taking oral contraceptives is 6-fold higher than that of non-users [16]. APLA and LA known to be prothrombotic in clinical
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manifestation were present in 5.8% cases. Hypertension was seen in 6.7% cases of which 2.9%
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cases also had a 4G/4G PAI-1 promoter polymorphism and high PAI-1 levels. Systemic infectious diseases like tuberculosis (2.9%) and typhoid (1.9%) along with polycythemia vera
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(1.9%) and recent trauma (16.3%) were some other acquired causes of CVT in these patients.
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ACCEPTED MANUSCRIPT Conclusion
Cerebral venous thrombosis is a multifactorial disease. Hereditary thrombophilia was seen in 16.3% of the cases. In addition to genetic, non-genetic and transient risk factors, dysfunctional fibrinolysis due to increased PAI-1 levels has been found to be strongly associated with CVT in
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the present study. Hypofibrinolysis with high PAI-1 levels mainly associated with PAI-1 4G/4G
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promoter polymorphism contributed to 19.2% of the CVT cases. Inflammation may also play a role in establishing an acquired state of hypercoagulabitity and hypofibrinolysis. Although
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genetic fibrinolytic disorders are rare, acquired cases of hypofibrinolysis seem to result in clinical manifestation of thrombosis. Combination of both heritable and acquired thrombophilia
fibrinolytic pathway will facilitate in a better
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markers both in the anticoagulant and
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along with hypofibrinolysis have a greater risk of CVT. Investigation of a combination of
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understanding of the pathophysiological mechanisms of cerebral venous thrombosis. Though CVT patients with abnormal levels of fibrinolytic and coagulation proteins were rechecked and
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reconfirmed yet the lack of a follow up study is a limitation of the study.
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ACCEPTED MANUSCRIPT Funding: This work is supported by the regular intramural funding of Indian Council of Medical Research (ICMR).
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Conflict of interest: None of the authors have conflict of interests to be declared.
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Authorship: The manuscript represents valid work. All authors certify that they have
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participated sufficiently in the work to take public responsibility for the content, including participation in the concept, design, analysis, writing, or revision of the manuscript. Furthermore,
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each author certifies that this material or similar material has not been and will not be submitted
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to or published in any other publication.
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Author contribution:
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BK, SS, KG conceived and designed the experiments. AP performed the experiments and analysed the data.
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AP, BK and SS wrote the paper.
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ACCEPTED MANUSCRIPT Figure Legend: Figure 1: Comparison of PAI-1 level with genotypes of PAI-4G/5G promoter polymorphism. There is a significant difference of PAI-1 (Plasminogen activator inhibitor-1) levels observed in
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4G/4G when compared to 4G/5G and 5G/5G genotypes (p=0.0103, Kruskal Wallis test).
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Figure 2: Scatter plots of laboratory values (thrombophilia and fibrinolysis parameters) of CVT
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patients in the present study.
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Table 1: Demographic and laboratory data of CVT cases in the present study 104 32.7 ± 11.8
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7 (6.7%) 4 (3.8%) 1 (1%) 5 (4.8%)
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64 (61.5%) 40 (38.5%)
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20 (19.2%) 19 (18.2%) 3 (2.9%) 2 (1.9%) 2 (1.9%)
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CVT cases Age (years) Sex Male Female Hereditary Thrombophilia PC deficiency (<60%) PS deficiency (<60%) AT deficiency (<60%) FVL mutation Hypofibrinolysis High PAI-1 (>110 ng/ml) PAI-1 4G/4G polymorphism Reduced tPA (<0.75 ng/ml) PLG deficiency (<300 µg/ml) High A2-AP (>200 ng/ml) Acquired or transient risk factors Hyperhomocysteinemia Trauma High CRP (>10 mg/L) Hypertension APLA/ LA Tuberculosis Typhoid Oral contraceptive use Pre-eclampsia History of pregnancy loss Polycythemia vera
20 (19.2%) 17 (16.3%) 14 (13.5%) 7 (6.7%) 6 (5.8%) 3 (2.9%) 2 (1.9%) 2 (5%)* 2 (5%)* 2 (5%)* 2 (1.9%)
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Abbreviations: PC: Protein C; PS- Protein S; AT: Antithrombin III, FVL: Factor V Leiden, PLGPlasminogen, PAI-1: Plasminogen activator inhibitor-1, tPA- Tissue plasminogen activator, A2-AP: Alpha-2-antiplasmin, APLA: Antiphospholipid antibodies, LA: Lupus Anticoagulant, CRP: C-reactive protein. * Risk factors in females alone (out of forty).
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ACCEPTED MANUSCRIPT Table 2: 5 CVT (4.8%) patients with combination of both hereditary and fibrinolysis markers Patient
Age (yr)
Sex
Combined Risk factors
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NU
SC
RI
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1 20 F PS deficiency, Reduced tPA, Oral contraceptive use 2 5 F PC deficiency, High PAI-1, PAI-1 4G/4G, High CRP 3 43 F PS deficiency, High PAI-1, PAI-1 4G/4G,Hyperhomocysteinemia 4 43 M FVL mutation, High PAI-1 5 36 M PC deficiency, High PAI-1, PAI-1 4G/4G and High A2-AP Abbreviation: PC: Protein C; PS- Protein S; FVL: Factor V Leiden, PAI-1: Plasminogen activator inhibitor-1, tPATissue plasminogen activator, A2-AP: Alpha-2-antiplasmin, CRP: C-reactive protein.
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ACCEPTED MANUSCRIPT Table 3: Comparison of the risk factors of the present study with other various studies Reference
[4]
[5]
[8]
[9]
[10]
[11]
[12]
[13]
Country
India
France
Tunisia
Poland
Italy
Turkey
United States
Iran
Number of patients
612
187
160
163
154
75
182
Study Design PC deficiency (%) PS deficiency (%) AT deficiency (%) FVL mutation (%)
R 8.5 4.8 2 3.1
P DN DN DN 8
R 1.8 4.3 1.8 DN
R 0 4 1 10
R 5.2 3.1 2.5 12.4
R 14.7 2 0 17.3
M 2 4 1 3
APLA/LA (%)
DN DN
11 DN
DN 7.5
11 1.8
21.5 4
Hyperhomocysteinemia (%)
DN
28
Infection (%)
6.5
16
DN 8.7
Trauma/ surgery (%)
-
5 5 10 57
1.8 3.7 31.8 23.5
PT gene G20210A (%)
4 14 12
D E
27 2
M
A
5
U N
C S
R 2.4 1.6 DN DN
13.3 DN
DN DN
DN 3.2
32 4
5 2
0.8 8.9
1
Pakistan
Germany
India
109
149
104
R 6 8 7 DN
P 4 5.4 3.4 14.8
P 6.7 3.8 1 4.8
DN 4
4.7 DN
DN 5.8
DN 29.5
19.2 1.9
29 18
9.67 9.4 16.3 Cancer (%) 2 10 7 Hematological causes (%) 1.6 1.9 OC use (%)* DN 34 71 5 46 3 5 Pregnancy/Postpartum/ adverse pregnancy 8 3 38.7 10 33.9 7 8.1 17 10 conditions(%)* Abbreviation: PC: Protein C, PS- Protein S, AT: Antithrombin III, FVL: Factor V Leiden, PT:Prothrombin, APLA: Antiphospholipid antibodies, LA: Lupus Anticoagulant, ACLA: Anticardiolipin antibodies, OC: Oral contraceptive, DN: Data not available , R- Retrospective, P- Prospective, M- Mixed * Risk factors in females alone
T P
E C
C A
20
4 10
Current study
T P
I R 124
[15]
[14]
ACCEPTED MANUSCRIPT
T P
I R
C S
A
U N
D E
M
T P
E C
C A Figure 1
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ACCEPTED MANUSCRIPT
T P
I R
C S
A
U N
D E
M
T P
E C
Figure 2
C A
22