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Abstract

Objective:

To evaluate the contribution of rs5918ITGB3 on the incidence and recurrence of deep venous thrombosis (DVT) in women and the relationship with body mass index (BMI) and smoking and to compare with data in men.

Results:

Rs5918(C) polymorphism in ITGB3 gene was assessed in 224 patients diagnosed with DVT and 216 controls. Thrombophilic genetic variant rs5918(C) was significantly pronounced in women (χ² =7.565, P = .008) and total patients (χ² = 9.266, P = .002) but not in men. Women patients (<45 years) who were carriers of rs5918ITGB3 polymorphism had an early onset of DVT (34.5 vs 39.4 years, χ² = 7.027, P = .008) as analyzed by Kaplan-Meier and a higher risk of the recurrent event (χ² = 3.405, odds ratio = 2.581, P = .044). The period before recurrent venous thromboembolism event was related to smoking status and BMI in young female who were carriers of rs5918 polymorphism but not in the males.

Conclusions:

Carriage of genetic variant rs5918(C) polymorphism in ITGB3 gene in women contributes to higher risk of single and recurrent DVT events at younger age.

Keywords

DVT glycoprotein IIb/IIIa/integrin B3 rs5918(C)/PlA1A2 recurrence early onset

Introduction

Deep venous thrombosis (DVT) is a rather common but preventable cause of morbidity and mortality worldwide. The annual incidence of venous thromboembolism (VTE) is around 0.117% in the general population¹ in a proportion of 1:1.2 (males:females). Incidence rates are relatively higher in women during childbearing years, whereas incidence rates are higher in men after the age of 45.²

The presence of multiple risk factors is a prerequisite for VTE development with synergistic gene–gene and gene–environment interactions, moderately increasing the risk above the sum of individual risk factors.³ Being a critical element of the clot-forming process, platelet glycoprotein (GP) IIb/IIIa (ITGB3) and its rs5918 (PlA1/A2) polymorphism were discussed as possible factors for prothrombotic tendency, along with commonly recognised inherited thrombophilic factors factor V Leiden (FVL) and prothrombin gene G20210A.^4,5

The PlA2 polymorphism is a thymidine-to-cytosine substitution at position 1565 in exon 2 (1565T>C) of the GPIIIa gene (17 chromosome, segment q21-q23) resulting in a leucine-to-proline substitution at a residue 33 of the β3 subunit of platelet GPIIb/IIIa.^6,7 This polymorphism is present in approximately 15% to 20% of the healthy population. The A2 allele of the platelet-specific alloantigen system is encoded by rs5918(C). It has been associated with the increased risk of myocardial infarction,⁸ heart disease,⁹ stroke,¹⁰ and resistance to blood-thinning benefits of aspirin.¹¹ It was also postulated as a risk factor for VTE,¹² with the prevalence in patients with VTE ranging from 14.5%¹³ to 31.6%.^14,15

The gender-related difference in platelet reactivity has been reported with the observed heightened responsiveness of platelets to thrombin in women, adenosine diphosphate (ADP), and binding more fibrinogen,^16,17 resulting in the formation of larger aggregates¹⁸ and more expressed activated GPIIb/IIIa receptors.¹⁹ A substantial gender difference with respect to the incidence and mortality of thrombosis-related diseases including VTE was reviewed by Middeldorp²⁰ and Naess et al.²¹ A hormonal exposure of women at the first event has been suggested to explain the difference in a risk observed in women as compared to men.²² There is still an array of redundancy and controversy in gender-related issues, particularly on the recurrence of VTE episodes.²³

The incidence of VTE is distributed unevenly over the age in patients with VTE: uncommon in young individuals and recognized as the leading cause of death in the elderly population.²⁴ However, recent studies have shown that PlA2 allele is a significant risk factor for acute coronary events in younger participants.^25,26 Although current options to diagnose DVT are well established, there are still some aspects of the risk of DVT regarding gender, age, recurrent issues, and complications that need to be addressed.

It is important to know whether the clinical features of DVT are similar in men and women, as well as to compare the prevalence of polymorphism carriership in men and women with suspected DVT. The causative roles for the thrombophilic conditions and their occurrence have not been investigated in depth. Data in the literature on platelet INGB3 (GPIIb/IIIa), its rs5918 (PlA1/A2) polymorphism, and relation to VTE are sparse and inconclusive. The aim of the study was to examine the association of rs5918 (PlA1/A2) polymorphism with clinical manifestation of DVT events in women as compared to men in subgroups younger than and older than 45 years.

Materials and Methods

Selection of Patients and Study Protocol

This prospective cohort study evaluated consecutive incidents of DVT and pulmonary embolism (PE) in patients of Caucasian origin, older than 18 years, treated in the clinic of Surgery at the University Hospital in Pleven, Bulgaria. A total of 224 unrelated patients with DVT were investigated (108 men and 116 women, aged 48.8 ± 15.05 years, age range: 18-79 years). The patients with DVT (311) were selected from 1821 consecutive patients (Supplementary flowchart). The selection criterion was a history of DVT with/without consequent PE. Deep venous thrombosis was diagnosed by compression, Doppler ultrasonography, and d-dimer measurements according to the criteria set by Evidence-Based Clinical Practice Guidelines.²⁷ From the 311 patients, those with malignant or myeloproliferative disease (n = 9, 2.9%), systemic inflammation (n = 3, 0.98%), advanced age (>80 years, n = 9, 2.9%), and no informed consent (n = 12, 3.9%), altogether 36 patients (11.6%), were excluded from the study group. The patients were followed up by regular visits every 6 months. Mean follow-up of the patients was 5.1 (0.5-10) years. Fifty-one (16.4%) patients were lost to follow-up. The patients at high risk and with recurrent events during follow-up were checked with Doppler ultrasound for venous status. Those with clinical signs of PE were additionally diagnosed with computed tomography pulmonary angiography and were treated at the University Pulmonology Clinics.

Treatment of Patients

Initial anticoagulation with low-molecular-weight heparin was applied for the first 5 days in patients diagnosed with DVT after assessment for bleeding risk according to commonly accepted recommendations.²⁸ In long-term anticoagulation for at least 6 months, oral anticoagulants were applied: indirect anticoagulant warfarin or, to avoid international normalized ratio monitoring, dabigatran or rivaroxaban. Patients contraindicated for warfarin received rivaroxaban. Only 2 patients did not receive long-term therapy, yet they did not have recurrent events during follow-up. The patients at high risk and with recurrent events were checked with Doppler ultrasound for the venous status and were treated with phlebotropic drugs twice a year for at least 3 months and 100 mg of aspirin daily.

Data Collection

A specific questionnaire was used for individual interviews with patients having thrombotic incidence (Supplementary material). The control group of 216 unrelated healthy individuals (108 men and 108 women, of age 45.92 ± 13.47 years) of Caucasian origin was selected from 282 volunteers with no personal VTE history, checked and confirmed by clinical examination at the department of surgery. The family history of DVT and VTE was taken from interviews. Twenty-three (8.2%) volunteers were excluded because of personal or family history of VTE, and 43 (15.2%) dropped out before the end of the study. Both patients and control groups were of similar demographic data and from the same geographical area. The data of demographic and baseline characteristics and common risk factors, collected by reviewing medical records, are presented in Table 1.

Table 1.

Clinical Parameters and Lifestyle Factors of the Patients With VTE According to Gender (Male and Female).^a

Characteristics	Total Patients, N (%)	Male Patients, N (%)	Female Patients, N (%)	P ^b
Patients	224	108 (52.72%)	116 (47.27%)
Age, years	48.8 ± 15.05	47.11 ± 14.97	49.22 ± 15.06	.05
Age of the first thrombotic incidence, years	46.42 ± 14.99	45.94 ± 15.12	46.88 ± 14.88	.05
Diagnosis of DVT	185 (84.4%)	92 (85.2%)	98 (84.1%)	.05
Thrombophlebitis/superficial thrombosis	36 (16.1%)	24 (22.2%)	12 (10.3%)	.05
Pulmonary embolism	35 (15.6)%	28 (25.90%)	7 (6.03%)	.05
Previous pulmonary embolism/deep vein thrombosis	70 (31.26%)	33 (30.55%)	37 (31.87%)	.05
Arterial thrombosis	4 (1.8%)	1 (0.9%)	3 (2.4%)	.05
Surgery	13.45%	14.8%	12.07%	.05
Trauma	14.73%	23.15%	7.76%	.05
Immobilization	13.07%	22.72%	5.1%	.05
Obesity	34.82%	37.03%	32.75%	.05
BMI, kg/m²	29.48	28.77	30.29	.05
Tobacco smoking ^c	38.46%	47.61%	28.03%	.05
Hormonal therapy/oral contraceptive (female patients)			7.76%
Pregnancy-related incident (female patients)			21.47%

Abbreviations: BMI, body mass index; DVT, deep venous thrombosis; VTE, venous thromboembolism.

^aValues are numbers and percentages in brackets.

^b P significance of the comparison of the data of male and female patients.

^cTobacco smoking status was defined as a current smoker or more than 10 years smoking in the past.

Body mass index (BMI) was calculated according to a formula: weight (kg) over height squared (m²). Blood clotting parameters were determined by conventional techniques in routine use at the University Hospital. The study protocol was approved by the Ethics Committee of Medical University-Pleven. All participants enrolled signed written informed consent for the investigation.

Sample Collection and DNA Analysis

The polymorphism rs5918 in INGB3 (PlA2 GPIIb/IIIa) was investigated by polymerase chain reaction (PCR). Venous blood was collected in vacutainers with 0.084 mL 15% ethylenediaminetetraacetic acid (Becton Dickinson and Company, Plymouth, UK). DNA was isolated following the procedure of GFTTM Genomic Blood DNA Purification Kit (Amersham Pharmacy Biotech Inc, Piscataway, NJ, USA) and quantified using the agarose gel procedure. The primers and reaction conditions for the investigated genetic defects have been described in detail in a previous publication.²⁹ Reagents from New England BioLabs Inc, Ipswich, MA, USA were used. The PCR guidelines were strictly followed to avoid contamination.³⁰

Statistical Analysis

Statistical analyses were performed using SPSS 21.0 for Windows (SPSS Inc, Chicago, Illinois). Continuous variables were expressed as means ± standard deviation. Categorical variables were expressed as percentages. The differences between continuous variables were analyzed using Student t-test, whereas those between categorical variables were analyzed using χ² test. Allele frequencies of investigated polymorphisms were calculated using a gene-counting method. Hardy-Weinberg equilibrium for the distribution of PlA1/A2 GPIIb/IIIa genotypes was tested by χ² test.

The prevalence was taken from cross-tabulations, and χ² estimates were calculated as Pearson χ² and a significance as Fisher exact test. Risk estimates were measured as odds ratio (OR) in 95% confidence intervals (CIs) with asymptomatic significance (2 sided) for the whole group of patients and compared to controls (with adjustment for sex and age) and stratified by gender.

Cumulative incidence of first and recurrent VTE events was estimated by the Kaplan-Meier survival method, censoring at the time of thrombotic event, at the event of recurrence, and at the end of follow-up.

The Kaplan-Meier analysis was used to measure the fraction of patients who were carriers of prothrombotic mutation and compared to noncarriers for “survival time” in anticipation of first VTE event as well as for survival time to the recurrent episode for estimating the survival function of the repeated event. The time interval was taken from medical records and follow-up data. The time interval for the patients who did not develop the recurrent episode was taken from follow-up history. Cox proportional hazard model was used to estimate the risk probability of VTE event occurring at younger age and associated with potential risk factors.

The correlations between different genotypes and recurrent event number as well as different genotypes and gender were studied in the groups stratified by gender and age. The cutoff age (45 years) was determined because of dramatic hormonal changes in females.³¹

Association between nonmodifiable risk factors, lifestyle variables, and DVT was evaluated with the χ² test or the Fisher exact test, and the relative risk for DVT was estimated. Recurrence of DVT, PE, and modifiable factors, including medical conditions and lifestyle factors, was presented as frequency distribution; surgery, trauma, immobilization, overweight, smoking habit (never vs sometimes), risk factors (no risk factors vs at least one) were all taken into account. Statistical significance was set at P < .05. The effect of the risk pattern was stratified for factors with significant differences between the DVT and the groups in the univariate model that comprised age, BMI, and recurrence.

Results

Clinical Parameters and Lifestyle Factors

The clinical parameters and lifestyle factors according to the gender of the assessed 224 patients are presented in Table 1. Women included in the study were insignificantly older than men. The total number of recurrent incidents was similar, and the number of PE episodes was less in women as compared to men (P = .03). Women had an additional risk such as a recent pregnancy or pregnancy-related incidents (21.47%) and using oral contraceptives or being on hormonal therapy (7.69%).

Carriage of Prothrombotic Mutation

The carriage of rs5918(C) in ITGB3 was significantly higher in female patients and the total group of patients as compared to controls but not in male patients (Table 2). The carriage of rs5918(C) polymorphism was calculated as a sum of prevalence of homozygous rs5918(C)/PlA2 and heterozygous carriage of rs5918(C/T)/PlA1/A2. Homozygosity is rather rare in the general population (less than 1%). None of the controls had it, but it was found in 2.87% of the patients (Supplementary Table 5). The calculated risk of DVT event was high in young females who were carriers of polymorphism (OR = 3.117, P = 0.011).

Table 2.

Prevalence, Pearson χ², Odds Ratio, Fisher Exact Test, and 95% CI of Rs5918(C) Polymorphism in ITGB3 in Male and Female Patients as Compared to Controls.

Statistical values	Prevalence in Patients, %	Prevalence in Controls, %	Pearson χ²	Fisher Exact Test (P)	Odds Ratio	95% CI
rs5918ITGB3/PlA1/A2	Prevalence in Patients, %	Prevalence in Controls, %	Pearson χ²	Fisher Exact Test (P)	Odds Ratio	95% CI
Female	36.7	20.20	7.565	.008^a	2.289	1.260-4.160
Male	28.4	18.1	2.541	.156	1.804	0.869-3.745
Total patients	32.8	19.30	9.266	.002^a	2.032	1.282-3.280
Female <45 years	37.3	17.6	7.393	.011^a	3.117	1.348-7.211
Male <45 years	22.0	16.0	0.898	.456	1.598	0.603-4.234
Total patients <45 years	29.1	16.8	6.391	.016^a	2.231	1.188-4.190

Abbreviation: CI, confidence interval.

^aSignificant P value.

According to Hardy-Weinberg, allele frequency was calculated as follows: female patients rs5918(C)/PlA244 (18.97%) and rs5918(T)/PlA1 188 (81.03%); male patients rs5918(C)/PlA2 32 (14.81%) and rs5918(T)/PlA1 184 (85.19%); and in controls: female controls 22 (10.81%) and 194 (89.19%); male controls 15 (7.08%) and 197 (92.9), respectively (Supplementary Table 5).

Kaplan-Meier Plots for DVT Event Probability

Kaplan-Meier plots for a probability of survival before DVT event in gender groups

By applying a Kaplan-Meier approach for statistical calculations, we analyzed how this polymorphism was related to the thrombotic event probability and its penetrance. The preliminary multifactorial analysis identified the following risk factors: carriage of polymorphism, age, sex, obesity, and smoking as significant or close to significance. Based on these results, the patients were divided into groups by gender and age and analyzed discretely by Kaplan-Meier for relationship of carriage/noncarriage with other factors. According to the guidelines on Kaplan-Meier statistics, “survival” times need not be related to actual survival, with death being a final event. However, an “event” may be any event of interest.³² In our case, Kaplan-Meier approach was used for the probability of survival before having DVT (Table 3, Figures 1 and 2, and Supplementary Table 6).

Table 3.

Means, χ², and Significance for 50% Probability of Event in Patients Younger Than and Older Than 45 Years, Carriers and Noncarriers of rs5918(C) Polymorphism in ITGB3 Gene.

Patients	rs5918(C)	Mean Estimate	Standard Error	95% CI	^aχ²	Fisher Exact Test (P)
Female <45 years	Noncarriers	39.366	0.892	37.618-39.366	7.027	.008^b
Female <45 years	Carriers	34.495	1.614	31.331-34.495
Male <45 years	Noncarriers	36.659	1.077	34.549-38.770	1.081	.298
Male <45 years	Carriers	34.516	2.343	29.923-39.109
Total patients <45 years	Noncarriers	37.876	0.692	36.520-39.232	6.424	.011^b
Total patients <45 years	Carriers	34.939	1.285	32.420-37.457
Female >45 years	Noncarriers	60.337	1.641	57.121-63.553	1.012	.314
Female >45 years	Carriers	62.367	2.443	57.579-67.156
Male >45 years	Noncarriers	59.578	1.531	56.577-62.580	0.637	.425
Male >45 years	Carriers	57.963	1.922	54.196-61.730
Total patients >45 years	Noncarriers	60.006	1.036	57.975-62.037	1.211	.292
Total patients >45 years	Carriers	61.498	1.691	58.183-64.813

Abbreviation: CI, confidence interval.

^aχ² log-rank Mantel-Cox.

^bSignificant P value.

Figure 1.

Survival time probability before DVT event for patients with deep venous thrombosis (DVT): (A) total patients (P = .023); (B) carriers of rs5918(C) in ITGB3 gene (P = .09); (C) female patients who were carriers and noncarriers of rs5918(C) in ITGB3 gene; and (D) male patients who were carriers and noncarriers of rs5918(C) in ITGB3 gene. From Kaplan-Meier probability curves, it is evident that the age-event tendency in younger and older groups is different in total patients (A) and in carriers and noncarriers (B), particularly in female patients (C).

Figure 2.

Survival time probability before deep venous thrombosis (DVT) event for the patients with DVT younger than 45 years (A, B, and C) and older than 45 years (D, E, and F), carriers and noncarriers of rs5918(C) in ITGB3 (GP IIb/IIIa) gene: (A) total patients, (B) female patients, (C) male patients, (D) total patients, (E) male patients, and (F) female patients.

The significant gender-related difference in DVT event probability was found for all the patients (χ2 = 5.190, P = .023; Figure 1A), and the mean age of the DVT event in females being higher than that of the males (Supplementary Table 6).

The mean age of DVT event (50% probability) was similar in each gender group for noncarriers and carriers of polymorphism. The difference in mean age of DVT event in the patients of both genders who are carriers of rs5918(C) polymorphism in ITGB3 gene was not significant, assumed because of the tendency in young patients opposite to this in the elderly patients, which was evident from Breslow analysis.

Kaplan-Meier plots for DVT event probability in age-related subgroups younger than and older than 45 years

The patients were divided into 2 age-related subgroups, younger than and older than 45 years (data of Breslow analysis). The significant difference in the probability of the early onset of the disease was found in the total group of patients younger than 45 years, who were carriers of polymorphism (P = .011), and a female subgroup (polymorphism carriers, younger than 45 years; P = .008; Figure 2A–C; Table 3). The mean estimates for the 50% probability of DVT event in young female patients were significantly different (39.5 vs 34.5 years) for carriers and noncarriers of PLA2, respectively (Table 3), but not in male patients.

No significant difference in the male group of patients who are carriers of rs5918 polymorphism in ITGB3 versus noncarriers was found. The patients >45 years, carriers of rs5918(C) polymorphism, had no significant difference in DVT event probability as compared to noncarriers (Figure 2D–F; Table 3).

Recurrence of Thrombotic Event

The recurrence of VTE event is one of the most important factors for clinicians to develop an adequate strategy for the treatment and prophylaxis. The recurrent events of previous DVT or PE were 31.26%, similar in male and female groups (30.55% and 31.87%, respectively; Table 1).

However, in patients who were carriers of rs5918ITGB3, the recurrence was significantly higher in women but not in men. Among women carriers of polymorphism, young women only were with significantly higher recurrent events (Table 4). However, the carriage of the same polymorphism in older women and men with recurrent events was not significantly different among the patients with recurrent events.

Table 4.

Genotype Frequency in the Patients With Recurrent Events, Carriers and Noncarriers of rs5918(C) ITGB3.^a

Statistical Values of Recurrent Events	Carriers of rs5918(C), %	Noncarriers of rs5918(C), %	Pearson χ²	OR	Fisher Exact Test (P)
Female <45 years	42.1	22.1	3.405	2.581	.044^b
Female >45 years	36.4	32.4	0.023	1.193	.723
Female	39.1	26.30	3.634	1.785	.025^b
Male <45 years	25.8	16.4	1.387	1.798	.239
Male >45 years	25.9	29.5	0.1345	0.782	.717
Male	25.9	22.6	0.518	1.266	.475

^aPrevalence, Pearson χ², and Fisher exact test.

^bSignificant P value.

The probability of the earlier recurrent event in young women who were carriers of polymorphism was higher but nonsignificant (χ2 Breslow = 2.683, P = .101). There was no difference in the period before the next event neither in older women nor in male patients with recurrent incident carriers and noncarriers of polymorphism (data not presented). Only 3 of the patients had recurrent events during anticoagulant therapy, and 2 of them were carriers of the investigated polymorphism.

Correlations Between Environmental Factors and rs5918(C) Polymorphism

Obesity and rs5918(C) polymorphism

Approximately 35% of the patients were obese and more than 50% were overweight. In obese female patients, the probability of DVT events was higher with later onset of the event (χ2 = 5.197, P = .023) as compared to nonobese female patients. In women who were carriers of the polymorphism, the effect was similar (χ2 = 3.678, P = .046; Figure 3; Supplementary Table 7). The probability of DVT event was significantly higher in young female patients who were carriers of rs5918(C) polymorphism but not significant in older women (χ2 = 3.592, P = .058; χ2 = 4.126, P = .177, respectively). There was no difference in male patients.

Figure 3.

Means, standard errors, and significance of 50% probability (A) of the first deep venous thrombosis (DVT) event and (B) of period (years) between first and second/recurrent event analyzed by Kaplan-Meier in female and male patients with DVT and obesity, carriers and noncarriers of rs5918(C) polymorphism in ITGB3 gene. *Significant values are presented.

The probability for a shorter period before the recurrent event in obese female patients who were carriers of rs5918(C) polymorphism was higher but not significant (χ2 = 2.371, P = .074). A significantly increased probability of the earlier recurrent event was found in all obese male patients (χ2 = 6.316, P = .012) and noncarriers of rs5918(C) polymorphism (χ2 = 5.722, P = .017; Figure 3; Supplementary Table 7). In obese male patients who were carriers of PlA2 polymorphism, the probability of DVT event had likely the same tendency. However, the χ² was low and insignificant.

Smoking and rs5918(C) polymorphism

In female patients who were smokers, a risk of DVT event was significantly higher (χ2 = 4.212, OR = 2.244, P = .040); however in male patients, the risk was not significantly higher than in nonsmokers (χ2 = 1.646, OR = 1.941, P = .200).

The probability of earlier DVT event in female patients who were smokers was significantly higher as compared nonsmokers (χ2 = 5.785, P = .016; Figure 4; Supplementary Table 8). In the carriers of rs5918(C)/PlA2 female patients, the effect was not significant.

Figure 4.

Means, standard errors, and significance of 50% probability (A) of the first deep venous thrombosis (DVT) event and (B) of period (years) between first and second/recurrent event in female and male patients with DVT, smokers, and nonsmokers, carriers and noncarriers of rs5918(C) polymorphism in ITGB3 gene analyzed by Kaplan-Meier. *Significant values are presented.

In female patients who were carriers of rs5918(C) polymorphism and smokers, the probability of the shorter period to the recurrent event (earlier onset of recurrent event) was significantly higher (χ2 = 4.679, P = .031) as compared to noncarriers (χ2 = 1.187, P = .666). A similar trend was noted in male patients carriers of rs5918(C) (χ2 = 5.423, P = .020) versus noncarriers (χ2 = 1.350, P = .245; Figure 4; Supplementary Table 8).

Discussion

The aim of the study was to evaluate the influence of thrombophilic rs5918 polymorphism in ITGB3 gene on the incidence and recurrence of DVT in women and to compare the results with those found in men, as well as the relationship of this polymorphism with BMI and smoking against the background of gender and age difference. Data and conclusions regarding the role of GPIIb/IIIa polymorphism in genetic susceptibility to thrombotic disease^7,10,12 in young and older males and females are controversial.^23,33,34

In the present study, the carriage of rs5918(C) polymorphism among female patients with DVT was shown to be significantly higher as compared to female controls, but no significant difference was found among the male patients. Furthermore, the 50% probability of the first DVT event in the female patients who were carriers of rs5918(C) was significantly higher as compared to the controls, particularly in the young female patients. This effect was seen neither in the male nor the older women.

These results reveal that carriage of rs5918(C) could be among the contributing risk factors for a single and recurrent DVT event in women at a young age. The probability of earlier DVT event in men and women younger than 45 years was found to be significantly different. The tendency in the probability of DVT in carriers of rs5918(C) polymorphism in ITGB3 gene at the younger age was higher in women than in men and was opposite later in the age. The higher risk of DVT in women as compared to men at the younger age has been seen in other publications²⁵ in relation to pregnancy and delivery or usage of oral anticonception.²⁴

Glycoprotein IIb/IIIa plays an essential role in the process of platelet aggregation and formation of thrombus plug.³⁵ Therefore, the association of rs5918(C) polymorphism was highly investigated, particularly in the incidence of coronary heart disease, and was presented in numerous studies by many investigators with ununiform results.^9

–16 Platelet function studies, as well as epidemiological findings on VTE, have often yielded contradictory results,³⁶ but overall, the polymorphism was found to be associated with increased platelet aggregability and platelet activation.³⁷

When investigating the role of rs5918 polymorphism in ITGB3, it is important to take into account that the carriers of rs5918(C) express higher reactivity in response to epinephrine, ADP activation,³⁷ enhanced thrombin generation, and shorter bleeding time.³⁸ What is more, platelets present gender-related difference in responsiveness that could be related to estrogen effects on the platelets.³⁹ The prothrombotic phenotype seen in these individuals could be the result of altered integrin signaling and cross-talking.

It has been shown that estradiol potentiates the activation of integrin B3 and the platelet aggregation induced by subthreshold concentrations of thrombin.³⁷ The expression of estrogen receptors both α and β in platelets is consistent with possible nongenomic effects of estrogen.^40,41 The activation of estrogen receptors is known to be linked with several downstream signaling pathways assembled into signalosome complex that includes G proteins, receptor tyrosine kinases, and nonreceptor tyrosine kinases such as Src⁴² and phosphatidylinositol kinase-3⁴³ related to induction of platelet effects.⁴⁴

The higher platelet number and reactivity in women,²² the higher expression of activated GPIIb/IIIa receptors, its vital role in blood clotting,^45,46 possible cross-talking of estrogen and ITGB3 receptors, and the data of higher penetrance of rs5918(C) in young female patients who were carriers of polymorphism could be explained by the aberration in the in–out signaling of the ITGB3 with the Leu33Pro amino acid substitution and the resulting conformational changes in the GP structure.⁴⁷ Upon summarizing this information, we could speculate that carriage of rs5918(C) polymorphism in ITGB3 gene (PlA1/A2) in young female patients appears to be associated with increased activation of platelets and increased prothrombotic activity.

Gender-related difference in the role of thrombophilic risk factors for recurrence of DVT was shown in the study conducted by Olié et al,²³ presenting a carriage of FVL polymorphism as the main factor for recurrence in men and obesity and aging in women. The present study is the first to investigate the contribution of carriage of prothrombotic polymorphism in platelet GP integrin B3 to higher risk and the early onset of DVT in gender-related groups.

Since in young women, carriers of rs5918(C) polymorphism, the risk of a recurrent event was found to be significantly higher than in noncarriers and this period of life is related to events with higher prothrombotic activities such as pregnancy, postpartum, and contraception, it is advisable to include this information in patient counseling.

Smoking

The relationship between smoking and VTE is important because smoking prevalence among men and women in many countries is still common. Smoking is a recognized risk factor for the thrombotic disease.⁴⁵ However, its role as an independent risk factor for VTE development remains unexplained. In our study, we found a significant relationship between smoking and DVT event and earlier onset of DVT in female patients. Nevertheless, the data on smoking and carriage of rs5918 polymorphism in male patients were inconsistent. However, both female and male patients, who were carriers of rs5918 polymorphism and smokers, had a shorter period before a recurrent event. In the literature, data on the relationship between smoking and risk of DVT are rather ambiguous. A recent meta-analysis showed a statistically insignificant OR for VTE of smokers as compared to nonsmokers,⁴⁸ whereas some prospective studies of Severinsen et al,⁴⁹ Hansson et al,⁵⁰ and Copenhagen study of Holst et al⁵¹ reported smoking to be an independent risk factor.

Assuming the multifactorial nature of VTE, we expected that the combined risk of smoking and polymorphism carriage would be synergistic and correlate with the findings of Pomp et al⁵² on the combined effect of FVL and current smoking leading to a 5.0-fold increased risk of DVT. Theoretically, knowing that smoking is associated with raised level of fibrinogen,^53,54 its enhanced interaction with the altered ITGB3 would lead to higher risk probability of DVT and possible recurrence. In the present study, this synergic effect of polymorphism carriage and smoking was found for the patients with recurrent events: higher recurrence and a shorter period to a recurrent event. However, this effect was not so pronounced for single DVT events.

Obesity

Our results show that obesity was related to the risk of DVT event, although later in life in both young and older female patients (data about age impact are not presented). In female carriers of rs5918(C) polymorphism, obesity was found to contribute significantly for the shorter period to a recurrent event. In obese male patients, the effect was seen in total patient group and the noncarriers. For the single DVT event, obesity contributed to later DVT event.

Recent studies have suggested that patients with obesity were at risk of developing VTE,^55,56 whereas some of the authors find the gender-related difference in the risk factors and the link with obesity in female patients only.²² Body mass index and waist-to-hip ratio have been shown to correlate positively with blood clot factors VII, VIIIc, fibrinogen, and Von Willebrand factor.^57,58 It might be a chronic inflammation related to obesity responsible for the observed elevations.⁵⁹

Glycoprotein ITGB3 is a receptor, and its extracellular part serves to bind activated platelets to ligands such as fibrinogen and Von Willebrand factor, facilitating platelet–platelet binding and platelet adhesion.⁶⁰ The binding of fibrinogen and Von Willebrand factor to ITGB3/GPIIb-IIIa is the final common pathway by which platelet aggregation occurs. Increased blood clot factors, particularly Von Willebrand factor and fibrinogen in obesity, could contribute to the higher activity of platelets and additionally altered responsiveness by ITGB3 (Leu33Pro) polymorphism, promoting venous thrombosis. The association of obesity and carriage of rs5918(C) polymorphism with VTE risk may be also mediated by the signaling of estrogen receptor and thus produce the gender-related effect of polymorphism carriage in an obese patient. A relationship between VTE obesity and ITGB3 may be possibly mediated by a variety of mechanisms.

Conclusion

The carriage of rs5918(C) in polymorphism in ITGB3 gene (PlA1/A2) was significantly higher among female patients with DVT as compared to women controls.

The probability of earlier onset of DVT event in young female patients, carriers of rs5918 polymorphism in ITGB3, was higher as compared to noncarriers.

The recurrent events were significantly higher in young women with DVT, carriers of rs5918 polymorphism in ITGB3, as compared to noncarriers and older female carriers.

The relationship between smoking and DVT event and earlier onset of DVT was found to be significant in female patients but not in the males. Female and male patients who carried rs5918 polymorphism and were smokers had a shorter period before a recurrent event.

Obese female patients, carriers of rs5918(C) polymorphism, showed a higher risk of DVT and a shorter period before the recurrent DVT event; in male patients, the effect was seen in noncarriers and the total patients.

The current study has some limitations. First, the number of the patients was limited, not sufficient to achieve significant values in obese and smoking patients’ subgroups, where OR was higher than 1.5. Second, we could not find any explanation of the delayed DVT onset in the obese female patients. Third, the retrospective assessment of “time interval” to recurrence was applied.

Thrombophilia testing remains a complex issue. The present study provides an evidence of gender-related particularities in the risk factors for single and recurrent VTE events.

The alteration in the risk for single and recurrent VTE in both men and women could hardly be elucidated by a single factor. Rather, it was the result from a combination of several risk factors. Further study is needed to investigate the responsiveness of platelets with the rs5918 polymorphism in ITGB3 and the mechanism of its interplay with the factors contributing to thrombosis.

The important reason to consider thrombophilia testing in a patient with idiopathic VTE is that the identification of thrombophilia factor could lead to a different approach in patient treatment and counseling or may require testing for the same factor in first-degree relatives.

Firstly, it is important to find out whether the inherited polymorphism contributes to the recurrent event. In our case, PlA2 contributed significantly to the recurrent event in young women only. Moreover, it contributed to an earlier onset of an event in young women. Therefore, we would recommend testing for this polymorphism in this subgroup of patients.

Secondly, it is important to consider what type of treatment would be recommended, at least it should be related to the reduction in the platelet reactivity. However, we do not suggest prolongation of anticoagulant therapy. Treatment strategies in this subgroup of patients could be the next step in our investigation.

Another important factor to consider is counseling the patients who are carriers of polymorphism on modifiable risks such as smoking and weight maintenance. Healthy, preventive lifestyle consistent with carriage of the polymorphism will prevent possible thrombotic events. Specific attitude to young women at a risk of a recurrent episode should be given, particularly in planning pregnancy or choice of contraceptives. Administration of optimal anticoagulation therapy and patient counseling should take into account carriage of platelet polymorphism.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Supplemental Material

The online [appendices/data supplements/etc] are available at .

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Rs5918ITGB3 Polymorphism,Smoking,and BMI as Risk Factors for Early Onset and Recurrence of DVT in Young Women

Abstract

Objective:

Results:

Conclusions:

Keywords

Introduction

Materials and Methods

Selection of Patients and Study Protocol

Treatment of Patients

Data Collection

Sample Collection and DNA Analysis

Statistical Analysis

Results

Clinical Parameters and Lifestyle Factors

Carriage of Prothrombotic Mutation

Kaplan-Meier Plots for DVT Event Probability

Kaplan-Meier plots for a probability of survival before DVT event in gender groups

Kaplan-Meier plots for DVT event probability in age-related subgroups younger than and older than 45 years

Recurrence of Thrombotic Event

Correlations Between Environmental Factors and rs5918(C) Polymorphism

Obesity and rs5918(C) polymorphism

Smoking and rs5918(C) polymorphism

Discussion

Smoking

Obesity

Conclusion

Footnotes

Declaration of Conflicting Interests

Funding

Supplemental Material

References