Discovery of glycyrrhetinic acid as an orally active,direct inhibitor of blood coagulation factor xa

Introduction

Factor Xa (FXa) plays an important role in blood coagulation. This study investigated glycyrrhetinic acid, a small molecule derived from Chinese herbs, and whether it has a direct inhibitory effect on FXa to display its anticoagulant activity.

Materials and Methods

Enzyme activities of FXa, plasmin, trypsin and thrombin, inhibition of FXa enzyme kinetics and plasma clotting time by glycyrrhentinic acid were performed in vitro. A rat tail-bleeding model and a rat venous stasis model were also used to evaluate in vivo tail-bleeding time and thrombus formation, respectively.

Results

Glycyrrhetinic acid in vitro directly inhibited FXa uncompetitivly with IC₅₀ of 32.6 ± 1.24 μmol/L, and displayed 2-, 14- and 20-fold selectivity for FXa when compared to plasmin, thrombin and trypsin, respectively. The plasma clotting time was increased in a dose-dependent manner. The prothrombin time doubled (PT₂), when the concentration of glycyrrhetinic acid reached 2.02 mmol/L. During in vivo experiments intragastric administration of glycyrrhetinic acid caused a dose-dependent reduction in thrombus weight on the rat venous stasis model (all P < 0.05). 50 mg/kg glycyrrhetinic acid resulted in 34.8% of venous thrombus weight lost, compared to the control. In addition, 200, 300 and 400 mg/kg doses of glycyrrhetinic acid caused a moderate hemorrhagic effect in the rat tail-bleeding model by prolonging bleeding time 1.1-, 1.5- and 1.9-fold compared to the control, respectively.

Conclusions

Glycyrrhetinic acid is a direct inhibitor of FXa that is effective by oral administration, and with further research could be used to treat blood coagulation disorders.     

Suppression of angiogenic response in local vein wall is associated with reduced thrombus resolution

Introduction

The formation of new vascular channels within and around venous thrombus contributes to its resolution. Neovascularisation arising from the surrounding vein may facilitate this process. Treatment of cancer patients with anti-angiogenic agents can lead to increased incidence of venous thromboembolic events, but the effect of these agents on the processes that govern thrombus resolution are unclear. The aim of this study was to determine the effect of anti-angiogenic treatment with 2-methoxyestradiol (2ME) on (i) angiogenic response in the thrombosed vein and (ii) venous thrombus resolution.

Materials and methods

Venous thrombus was induced in the inferior vena cava (IVC) of 36 adult male BALB/C mice. Thrombosed mice received either the anti-angiogenic agent, 2ME (150 mg/kg/day, i/p), or vehicle control (n = 18/group). In the thrombosed IVC of both groups: hypoxia-inducible factor (HIF) 1α, and its angiogenic targets, vascular endothelial growth factor (VEGF) and placental growth factor (PLGF), were quantified using enzyme-linked immunosorbent assays at days 1 and 10 post-thrombus induction (n = 6/group); and inflammatory cell content, cell proliferation, and vein recanalisation were quantified using immunostaining and image analysis at day 10 (n = 6/group).

Results

In the IVC of mice treated with 2ME compared with control: HIF1α (P < 0.005 and P < 0.02), VEGF (P < 0.005 and P < 0.02), and PLGF levels (P < 0.01 and P < 0.001) were reduced at days 1 and 10 post-thrombus induction respectively, and macrophage content (P < 0.005), neutrophil content (P < 0.01), vein recanalistion (P < 0.05), and thrombus resolution (P < 0.001) were also reduced at day 10.

Conclusions

Anti-angiogenic treatment with 2ME supressed the HIF1-mediated angiogenic drive in local vein wall and attenuated venous thrombus resolution. The potential pro-thrombotic effect of anti-angiogenic agents should be carefully considered when managing venous thromboembolic events in cancer patients.     

Genetic variants in Cell Adhesion Molecule 1 (CADM1): A validation study of a novel endothelial cell venous thrombosis risk factor

Introduction

In a protein C deficient family, we recently identified a candidate gene, CADM1, which interacted with protein C deficiency in increasing the risk of venous thrombosis (VT). This study aimed to determine whether CADM1 variants also interact with protein C pathway abnormalities in increasing VT risk outside this family.

Materials and methods

We genotyped over 300 CADM1 variants in the population-based MEGA case-control study. We compared VT risks between cases with low protein C activity (n = 194), low protein S levels (n = 23), high factor VIII activity (n = 165) or factor V Leiden carriers (n = 580), and all 4004 controls. Positive associations were repeated in all 3496 cases and 4004 controls.

Results

We found 22 variants which were associated with VT in one of the protein C pathway risk groups. After mutual adjustment, six variants remained associated with VT. The strongest evidence was found for rs220842 and rs11608105. For rs220842, the odds ratio (OR) for VT was 3.2 (95% CI 1.2-9.0) for cases with high factor VIII activity compared with controls. In addition, this variant was associated with an increased risk of VT in the overall study population (OR: 1.5, 95% CI 1.0-2.2). The other variant, rs11608105, was not associated with VT in the overall study population (OR: 1.0, 95% CI 0.8-1.1), but showed a strong effect on VT risk (OR: 21, 95% CI 5.1-88) when combined with low protein C or S levels.

Conclusions

In a population-based association study, we confirm a role for CADM1 variants in increasing the risk of VT by interaction with protein C pathway abnormalities.     

Recanalization of occluded large arteries with broadened therapeutic window for acute cerebral infarction

Objective

To investigate the safety and efficacy of recanalization with a broadened therapeutic window for acute occlusion of large cerebral arteries.

Methods

We assessed 38 patients who underwent the hyper-selective intra-arterial administration of low-dose urokinase, along with mechanical thrombus disruption or mechanical thrombus disruption recanalization (34 stents in 33 patients) of occluded vessels, 20 with onset time-to-treatment (OTT) >6 h (observation group; mean OTT, 20.10 ± 15.67 h) and 18 with OTT ≤6 h (control group). NIHSS scores, mRS scores (≤2) at 3 months, recanalization rates, severe cerebral infarctions on CT, and symptomatic hemorrhagic conversions after surgery were compared.

Results

Postoperative recanalization rates were 100% in both groups, and other results were equivalent.

Conclusions

Recanalization at longer OTT was safe and effective with acute occlusions of large cerebral arteries. Time to recanalization could be safely prolonged for up to 20 h in these patients.     

New method of thrombus preparation using a fluid model for evaluation of thrombectomy devices in a swine model

Background

Mechanical thrombectomy is a promising new modality of interventional stroke treatment. Preparation of thrombus is a very important step for the evaluation of the mechanical thrombectomy devices. The objective of this study was to explore a new method of thrombus preparation with fluid model (FM) for assessment of thrombectomy devices used in the recanalization of acute ischemic stroke.

Methods

Elongation test and catheter injection test were used to evaluate the mechanical properties of thrombi prepared by FM and static model (SM). Histological structures of two artificial clots and specimens of stroke patients were compared. Radiopacity of thrombus made by FM was evaluated in a swine embolization model.

Results

The maximum tensile length of thrombi prepared by FM and SM were significantly higher (4.28 ± 0.23 cm vs 3.16 ± 0.13 cm, P < 0.01) and showed less breakage on catheter injection test (13% vs 60%, P < 0.05). Histological features of thrombi prepared by FM showed mixed thrombus structure, similar to thromboemboli retrieved from acute stroke patients, while clots generated by SM were replete with erythrocytes. A total of twelve vessels in two swine were successfully occluded (TIMI 0 or 1), with sufficient radiopacity of each injected thrombus.

Conclusion

The thrombus prepared by FM had good mechanical stability, sufficient radiopacity, and similar histological structure of thromboemboli retrieved from stroke patients, which make it possible to be used in the evaluation of thrombectomy devices.     

Antithrombotic effects of a newly purified fibrinolytic protease from Urechis unicinctus

Introduction

The prevalence of thromboembolic disease, one of the top 3 leading causes of mortality worldwide, is being reported continually. More effective and safer antithrombotic drugs may overcome the underlying problems in antithrombotic therapy. In the present work, antithrombotic effects of UFEIII, a newly purified fibrinolytic protease from Urechis unicinctus were evaluated.

Materials and methods

UFEIII was purified from the marine invertebrate, Urechis unicinctus, using anion exchange and gel filtration chromatography. Molecular weight, fibrinolytic activity and fibrinogenolysis pattern of UFEIII were determined. Furthermore, antithrombotic effects of UFEIII in vivo were investigated through electrical induced carotid arterial thrombosis in rats, FeCl₃ induced carotid arterial thrombus model in rabbits and stasis induced vena caval thrombus model in rats.

Results

SDS-PAGE of the purified enzyme showed a single polypeptide chain with molecular weight of 20.8 kDa. In fibrin plate assays, UFEIII could not only directly degrade fibrin but also activate plasminogen. The fibrinogenolysis pattern of UFEIII was Aα-chains > Bβ-chains > γ-chain. Moreover, UFEIII could effectively prolong the time to occlusion in electrical induced carotid arterial thrombosis. Besides, both in rabbits and rats, the administration of UFEIII not only prolonged the activated partial thromboplastin time (APTT) and thrombin time (TT) also decreased the fibrinogen (FIB) content. Further, the thrombus lysis was observed after administration of UFEIII both in rabbits and rats.

Conclusion

UFEIII can possibly be a new potential source of fibrinolytic agent.     

Urokinase plasminogen activator independent early experimental thrombus resolution: MMP2 as an alternative mechanism

Deep-vein thrombosis (DVT) resolution is thought to be primarily a urokinase plasminogen activator (uPA) -dependent mechanism, although observations suggest other non-fibrinolytic mechanisms may exist. We explored the role of matrix metalloproteinase (MMP) -2 and -9 in early DVT resolution in uPA-deficient mice. Male B6/SVEV (WT) and genetically matched uPA -/- mice underwent inferior vena cava (IVC) ligation to create stasis venous thrombi, with IVC and thrombus harvest. Thrombus size was similar between WT and uPA -/- mice at day 4, suggesting early non uPA-dependent resolution. Intrathrombus neutrophils and monocytes were reduced 3- and 3.5-fold in uPA -/- mice as compared with WT. By ELISA, tumour necrosis factor α and interleukin 1β were not altered, while interferon (IFN)γ was significantly elevated in uPA -/- mice. A compensatory increase in thrombus tPA was not observed, plasmin activity was reduced and PAI-1 was elevated 2.5-fold in uPA -/- mice. Active MMP2, but not MMP9, was elevated 3-fold in uPA -/- mice as compared with WT as well as MMP-14, an MMP2 activator. Collagen type IV and fibrinogen were reduced in uPA -/- mice thrombi as compared with WT. IFNγ induces MMP2, and blockade of IFNγ was associated with larger venous thrombi and reduced active MMP2, as compared with WT. Consistently, MMP2 -/- mice had larger VT as compared with WT controls, despite normal thrombus plasmin levels. Taken together, early experimental venous thrombus resolution is independent of uPA, and, in part, inflammatory cell influx. MMP2-dependent thrombolysis is an important compensatory mechanism of venous thrombus resolution, possibly by collagen type IV metabolism, and may represent an exploitable therapeutic avenue.     

Inhibition of factor XI reduces thrombus formation in rabbit jugular vein under endothelial denudation and/or blood stasis

Introduction

In addition to acquired and inherited risk factors, the growth of venous thrombus under static conditions and endothelial injury play important roles in the development of deep venous thrombosis (DVT), for which risk factors include increased plasma levels of coagulation factor XI (FXI). The aim of this study is to understand the role of FXI in venous thrombus formation under conditions of endothelial denudation and/or blood stasis.

Materials and methods

The contribution of FXI to venous thrombus formation was investigated in a rabbit model and a flow chamber system. Thrombi were induced in the rabbit jugular veins by (1) endothelial denudation, (2) vessel ligation (blood stasis) or (3) by combined endothelial denudation and vessel ligation. Blood samples were perfused on immobilized type III collagen at a wall shear rate of 70/s and then the surface area covered by platelets and fibrin was morphometrically evaluated. Prothrombin fragment 1 + 2 (F1 + 2) generation was also measured before and after perfusion.

Results

All thrombi induced in rabbit jugular veins were composed of platelets, fibrin and erythrocytes. Anti-FXI antibody significantly reduced ex vivo plasma thrombin generation initiated by ellagic acid but not by tissue factor, and in vivo thrombus formation under endothelial denudation and/or vessel ligation. The antibody significantly reduced surface areas covered by platelets and fibrin, as well as F1 + 2 generation at a wall shear rate of 70/s in flow chambers.

Conclusion

These results suggest that FXI contributes to venous thrombus growth under conditions of endothelial denudation and/or blood stasis, and that thrombin generation by FXI interaction promotes further platelet aggregation and fibrin formation at low shear rates.     

Differences in thrombus structure and kinetics in patients with type 2 diabetes mellitus after non ST elevation acute coronary syndrome

Introduction

Despite optimal secondary prevention therapy following non-ST elevation acute coronary syndrome (NSTE-ACS), recurrent thrombotic events are more frequent in patients with type 2 diabetes mellitus (T2DM).

Materials and Methods

This exploratory study was aimed to evaluate quantitative and qualitative aspects of thrombus. In 28 patients with and without T2DM treated with aspirin and clopidogrel we assessed thrombus quantity using an ex-vivo chamber, platelet reactivity, thrombus ultrastructure and thrombus kinetics one week after NSTE-ACS.

Results

T2DM was associated with increased thrombus [14861 (8003 to 30161) vs 8908 (6812 to 11996), μ²/mm, median (IQR), p = 0.045] and platelet reactivity. In addition, diabetic thrombus showed lower visco-elastic tensile strength [(− 0.2(− 1.7 to 0.7) vs 1.0(− 0.9 to 3.3), p = 0.044)] and was more resistant to autolysis [(27.8(11.7 to 70.7) vs 78.8(68.5 to109.6) mm/min, p = 0.002)]. On SEM, fibrin fibres in diabetes were thinner, with higher lateral interlinkage and mesh-like organisation. Thrombus quantity correlated inversely with thrombus retraction (r = − 0.450 p = 0.016) but not with platelet reactivity (r = 0.153, p = 0.544).

Conclusions

Despite optimal antiplatelet therapy, T2DM patients after NSTE-ACS developed increased thrombus of lower tensile strength and slower retraction. SEM revealed loosely arranged fibrin fibres. Our data showed significant differences in the magnitude as well as structural and mechanistic characteristics of thrombus in patients with T2DM.     

Paraoxonase-1 activity affects the clopidogrel response in CYP2C19 loss-of-function carriers

Background

The impact of paraoxonase-1 (PON1) activity on the response to clopidogrel may differ in patients treated with drug-eluting stents (DES) in association with CYP2C19 loss-of-function (LOF) polymorphisms.

Methods

This study included 112 Japanese patients receiving clopidogrel (75 mg/day) and aspirin (100 mg/day) who underwent optical coherence tomography (OCT) examination 9 months after DES implantation. The CYP2C19 genotype was analyzed and LOF carriers (*1/*2, *1/*3, *2/*2, *3/*3, *2/*3) were identified. At the 9-month follow-up, platelet reactivity was determined by measuring the P2Y12 reactivity unit (PRU) using a VerifyNow P2Y12 assay, PON1 activity was evaluated and intra-stent thrombus was evaluated by OCT.

Results

Of the 112 Japanese patients, 75 were LOF carriers (67.0%). The patients were divided into tertiles according to the PON1 activity (tertile 1; < 230 U/L, tertile 2; 230–283 U/L, tertile 3; > 283 U/L). In the VerifyNowP2Y12 analysis, tertile 1 had a higher PRU than tertiles 2 and 3 in LOF carriers, and there was no difference among tertiles in non-carriers. The highest incidence of intra-stent thrombus was observed in tertile 1 followed by tertiles 2 and 3 in LOF carriers, whereas there was no such difference in non-carriers. Multivariate analysis revealed that LOF carriers and PON1 activity tertile 1 were independent predictors of intra-stent thrombus in all patients. In LOF carriers, tertile 1 was the only independent predictor for intra-stent thrombus.

Conclusion

Low PON1 activity is associated with a low response to clopidogrel and a high frequency of intra-stent thrombus only in LOF carriers.     

Bivalirudin is inferior to heparin in preservation of intraoperative autologous blood

Introduction

Bivalirudin is used as an alternative to heparin in cardiac surgery, and may be superior to heparin with regard to platelet function. Bivalirudin however, is prone to cleavage by thrombin resulting in coagulation in areas of stasis.

Material and Methods

We compared the preservation of platelet function and the quality of anticoagulation in autologous blood of 26 cardiac surgical patients collected intraoperatively and anticoagulated ex vivo with either bivalirudin or heparin, with supplementation of bivalirudin over time and prevention of stasis.

Results

We found in both preservatives a reduction in ADP-induced platelet aggregation response over a period of 105 minutes (median, IQR: 73-141) as measured by Multiplate®. Supplementation of additional bivalirudin (23 ± 1.1 μg/ml/hr) and prevention of stasis was not able to prevent thrombin generation. We found a 5-fold increase in levels of prothrombin fragment 1 + 2 in bivalirudin preserved autologous blood as compared to heparin preserved blood (F₁₊₂ levels median 8.9 nM [quartile percentiles 4.2-12.4] vs 1.3 nM [0.6-2.1], P = 0.001 Mann-Whitney, n = 10).

Conclusions

Our study suggests that preservation of platelet function in autologous blood anticoagulated with bivalirudin is not a suitable alternative to heparin.     

Risk factors for recurrent venous thromboembolism in young and middle-aged women

Background

It is a matter of debate whether women with an episode of VTE associated with estrogen have a lower risk of recurrence than women with an unprovoked VTE.

Objectives

To identify risk factors for recurrent VTE in women and to assess the risk of recurrent VTE associated with combined oral contraceptives (CHC) or menopausal hormone treatment (HT), compared to surgery-related and unprovoked VTE.

Patients/Methods

A cohort of 974 women aged 18–64 years with a first episode of VTE were followed-up for a median time of 5.2 years. All women were previously included as cases in the Swedish nation-wide case–control study “Thrombo Embolism Hormone Study” (TEHS). Hazard ratios for recurrence were calculated using univariable and multivariable Cox proportional hazards model.

Results

A total of 102 patients (10%) suffered from recurrent VTE. The annual rate of recurrence was 1.0% in patients with surgery/cast, 2.0% in patients with CHC/HT and 3.2% in patients with unprovoked first VTE. Adjusted hazards ratio (HR_a) for recurrence was 0.35 (95% CI 0.20-0.61) in women with VT provoked by surgery/cast while women with estrogen-associated VTE had a HR_a of 0.70 (95% CI 0.43-1.20) compared to women with unprovoked VTE.

Conclusion

Women 18–64 years are at low risk of recurrent VTE. Women with hormone associated VTE had a lower risk of recurrence than women with unprovoked VTE, but not as low as surgery/cast provoked VTE.     

Myeloid cell tissue factor does not contribute to venous thrombogenesis in an electrolytic injury model

Introduction

Tissue factor (TF) is a potent initiator of the extrinsic coagulation cascade. The role and source of TF in venous thrombotic disease is not clearly defined. Our study objective was to identify the contribution of myeloid cell TF to venous thrombogenesis in mice.

Materials and methods

The mouse electrolytic inferior vena cava model was used to induce thrombosis. The following groups of mice were used (1) TF^flox/floxLysMCre⁺ mice that have reduced TF expression in myeloid cells, (2) TF^flox/floxLysMCre^- littermate controls, (3) Wild type mice given a monoclonal anti-mouse TF antibody (1H1) to inhibit TF activity, and (4) Wild type mice given rat IgG. Evaluations at baseline, day 2, and day 6 post thrombosis included thrombus weight, vein wall inflammatory cell migration, vein wall TF mRNA, and plasma D-dimer levels.

Results

Inhibition of TF significantly decreased thrombus weight 2 days post venous thrombosis. In contrast, TF^flox/floxLysMCre⁺ had no change in thrombus weight when compared to littermate controls. The absence of myeloid cell TF did not affect infiltration of neutrophils or monocytes into the vein wall. TF mRNA expression in the vein wall decreased at 2 days but then returned to baseline levels by 6 days post thrombosis. D-dimer levels peaked at 2 days post thrombosis in mice with or without myeloid cell TF.

Conclusions

TF is important in the formation of venous thrombi in the macrovasculature. However, TF expression by myeloid cells does not significantly contribute to venous thrombogenesis in this model.     

Association between obstructive sleep apnea and deep vein thrombosis / pulmonary embolism: A population-based retrospective cohort study

Background

Obstructive sleep apnea (OSA) is a major contributor to cardiovascular disease, and may cause severe morbidity and mortality. Recent studies have indicated that OSA patients exhibited elevated platelet activity, fibrinogen levels, and platelet aggregation.

Objectives

We investigated the risk of deep vein thrombosis (DVT) and pulmonary embolism (PE) in patients diagnosed with OSA compared with age- and sex-matched unaffected people.

Patients/Methods

This longitudinal, nationwide, population-based cohort study was conducted using data from Taiwan National Health Insurance Research Database (NHIRD) recorded between January 2000 and December 2011. The study consisted of 3511 patients with OSA and 35110 matched comparison individuals. A Cox proportional hazard regression was used to compute the risk of DVT and PE in patients with OSA compared with those without OSA.

Results

The DVT and PE risks were 3.50- and 3.97-fold higher (95% CI = 1.83–6.69 and 1.85–8.51) respectively, in the OSA cohort than in the reference cohort after we adjusted for age, sex, and comorbidities.

Conclusion

This nationwide population-based cohort study indicates that patients with OSA exhibit a higher risk of subsequent DVT and PE.     

Open-label,randomized study of the effect of rivaroxaban with or without acetylsalicylic acid on thrombus formation in a perfusion chamber

Introduction

Rivaroxaban, a direct factor Xa inhibitor, has demonstrated effectiveness for the management of both venous and arterial thrombosis. This study was designed to investigate the antithrombotic effect of rivaroxaban, with or without acetylsalicylic acid (ASA), in an ex vivo perfusion chamber at both low and high shear rates.

Materials and methods

Healthy subjects (N = 51) were enrolled in a randomized, crossover (rivaroxaban 5, 10 or 20 mg with or without ASA), and parallel-group (compared with ASA plus clopidogrel) study. Thrombi formed on pig aorta strips were measured after a 5-minute perfusion at low and high shear rates with blood from the subjects by measuring D-dimer concentration (for fibrin deposition) and P-selectin content (for platelet deposition).

Results

ASA alone had no impact on thrombus D-dimer levels, whereas rivaroxaban alone at peak concentrations decreased D-dimer levels by 9%, 84% and 65% at low shear rate and 37%, 73% and 74% at high shear rate after doses of 5, 10 and 20 mg, respectively. Steady-state ASA plus rivaroxaban 5 mg caused a greater reduction in D­dimer levels (63%) than monotherapy at low shear rate. Co-administration of ASA with clopidogrel was associated with a 30% decrease in D-dimer levels at low shear rate and a 14% decrease at high shear rate. No conclusive effect on P-selectin content was observed across the treatment groups.

Conclusions

Rivaroxaban dose-dependently inhibited ex vivo thrombus formation under low and high shear rates. Co-administration of ASA had an additional effect on the antithrombotic action of low-dose rivaroxaban.     

Comparison of lumbar drainage and external ventricular drainage for clearance of subarachnoid clots after Guglielmi detachable coil embolization for aneurysmal subarachnoid hemorrhage

Objective

Subarachnoid clots play an important role in development of delayed vasospasm after subarachnoid hemorrhage (SAH). The purpose of this study was to compare clearance of subarachnoid clots using external ventricular drainage (EVD) or lumbar drainage (LD) after Guglielmi detachable coil (GDC) embolization for aneurysmal SAH.

Methods

The subjects were 51 treated with GDC coil embolization for aneurysmal Fisher group 3 SAH within 72 h of ictus. Software-based volumetric quantification of the subarachnoid clots was performed on CT scans and the hemoglobin (Hb) level was measured in CSF drained from each catheter.

Results

Clearance of subarachnoid clots was more rapid in patients treated with LD (n = 34) compared to those treated with EVD (n = 17). The Hb level in CSF was significantly higher in the LD group on Days 4–5 after onset of SAH (P < 0.05), but was higher in the EVD group on Days 8–9. The incidence of symptomatic vasospasm did not differ between the two groups. The rate of occurrence of a new low density area on CT scans was higher in patients treated with EVD, but not significantly higher than the rate in the LD group.

Conclusion

GDC embolization followed by lumbar drainage accelerates the reduction of subarachnoid clots, but EVD may contribute to stasis of hemorrhage within subarachnoid spaces.     

Enhanced thrombin generation in women with a history of oral contraception-related venous thrombosis

Introduction

In women who suffer venous thrombosis (VT) during oral contraceptive (OC) use, a transient risk factor (OC) is removed during the acute event, while most co-existing forms of thrombophilia persist and presumably continue to maintain hypercoagulability. The aim of this study was to establish if hypercoagulability persists long after OC-related VT and if it could be attributed to thrombophilia.

Materials and Methods

60 women (age 33.0 ± 8.5 years) were investigated 5 – 64 (median 33) months after OC-related VT (patients) and compared to 63 apparently healthy women (controls). All women were tested for thrombophilia, activated partial thromboplastin time (APTT), fibrinogen, D-dimer, P-selectin and C-reactive protein. Thrombin generation was measured by Technothrombin® TGA assay. Overall haemostasis potential (OHP) assay with overall coagulation potential (OCP) and overall fibrinolytic potential (OFP) as supplementary parameters were measured by repeated fibrin formation and degradation registration.

Results

In patients increased endogenous thrombin potential (4205 ± 440 nM x min vs 4015 ± 421 nM x min, p = 0.017), increased OCP (22.6 ± 4.6 Abs-sum vs 20.8 ± 4.1 Abs-sum, p = 0.025), shorter APTT (30.9 ± 3.8 s vs 33.4 ± 3.6 s, p < 0.001) and lower antithrombin activity (99, 93-105% vs 104, 100-109%, p < 0.05) were observed. Thrombophilia was observed in 22/60 (36%) patients and in 5/63 (7.9%, p < 0.001) controls. The only significant difference between thrombophilic and non-thrombophilic patients was higher soluble P-selectin in the former subgroup (22, 20-33 μg/L vs 17, 12-22 μg/L, p = 0.012).

Conclusions

In women with a history of OC-related VT persistent hypercoagulability was observed, which, however was not augmented by the presence of thrombophilia.     

Thrombotic safety of prothrombin complex concentrate (Beriplex P/N) for dabigatran reversal in a rabbit model

Introduction

In vivo animal data have shown prothrombin complex concentrate (PCC) to be effective in preventing bleeding induced by excessive plasma levels of the direct thrombin inhibitor dabigatran. This animal model study was designed to determine the risk of thrombosis associated with administration of a PCC (Beriplex P/N) to reverse dabigatran-induced bleeding.

Materials and methods

Anesthetized rabbits were treated with initial 0, 75, 200 or 450 μg kg^- 1 dabigatran boluses followed by continuous infusions to maintain elevated plasma dabigatran levels. At 15 min after the start of dabigatran administration, PCC doses of 0, 50 or 300 IU kg^- 1 were administered. Thereafter, coagulation in an arteriovenous (AV) shunt was evaluated and histopathologic examination for thrombotic changes performed. Venous thrombosis was also assessed in a modified Wessler model.

Results

At the suprapharmacologic dose of 300 IU kg^- 1, PCC increased thrombus weight during AV shunting, but this effect could be prevented by dabigatran at all tested doses. AV shunt occlusion after PCC administration was delayed by 75 μg kg^- 1 dabigatran and abolished by progressively higher dabigatran doses. High-dose treatment with 300 IU kg^- 1 PCC resulted in histologically evident low-grade pulmonary thrombi; however, that effect could be blocked by dabigatran in a dose-dependent manner (p = 0.034). In rabbits treated with high-dose PCC, dabigatran inhibited thrombus formation during venous stasis. PCC effectively reversed dabigatran-induced bleeding.

Conclusions

In this animal study, thrombosis after PCC administration could be prevented in the presence of dabigatran. PCC reversed dabigatran-induced excessive bleeding while retaining protective anticoagulatory activity of dabigatran.     

The Methylenetetrahydrofolate Reductase Polymorphism (MTHFR c.677C > T) and Elevated Plasma Homocysteine Levels in a U.S. Pediatric Population with Incident Thromboembolism

Objective

Elevated plasma homocysteine (tHcy) and the MTHFR c.677C > T variant have been postulated to increase the risk of venous thromboembolism (VTE), although mechanisms and implications to pediatrics remain incompletely understood. The objectives of this study were to determine the prevalences of elevated tHcy and MTHFR variant in a pediatric population with VTE or arterial ischemic stroke (AIS), and to determine associations with thrombus outcomes.

Study Design

Subjects were enrolled in an institution-based prospective cohort of children with VTE or AIS. Inclusion criteria consisted of objectively confirmed thrombus, ≤ 21 years at diagnosis, tHcy measured and MTHFR c.677C > T mutation analysis. Clinical and laboratory data were collected. Frequencies for elevated tHcy and MTHFR variant were compared with NHANES values for healthy US children and also between study groups (VTE vs AIS, provoked vs idiopathic) and by age.

Results

The prevalences of hyperhomocysteinemia or MTHFR variant were not increased in comparison to NHANES. tHcy did not differ between those with wild-type MTHFR versus either c.677C > T heterozygotes or homozygotes. There was no association between tHcy or MTHFR variant and thrombus outcomes.

Conclusion

In this cohort of US children with VTE or AIS, neither the prevalence of hyperhomocysteinemia nor that of MTHFR variant was increased relative to reference values, and adverse thrombus outcomes were not definitively associated with either. While it is important to consider that milder forms of pyridoxine-responsive classical homocystinuria will be detected only by tHcy, we suggest that routine testing of MTHFR c.677C > T genotype as part of a thrombophilia evaluation in children with incident thromboembolism is not warranted until larger studies have been performed in order to establish or refute a link between MTHFR and adverse outcomes.     

Antithrombin deficiency in three Japanese families: One novel and two reported point mutations in the antithrombin gene

Introduction

Inherited antithrombin (AT) deficiency is associated with a predisposition to familial venous thromboembolic disease. We analyzed the AT gene in three unrelated patients with an AT deficiency who developed thrombosis.

Materials and Methods

We analyzed the SERPINC1 gene in three patients. Additionally, we expressed the three mutants in the COS-1 cells and compared their secretion rates and levels of AT activity with those of the wild-type (WT).

Results

We identified three distinct heterozygous mutations of c.2534C>T: p.56Arginine → Cysteine (R56C), c.13398C>A: p.459Alanine → Aspartic acid (A459D) and c.2703C>G: p.112 Proline → Arginine (P112R). In the in vitro expression experiments, the AT antigen levels in the conditioned media (CM) of the R56C mutant were nearly equal to those of WT. In contrast, the AT antigen levels in the CM of the A459D and P112R mutants were significantly decreased. The AT activity of R56C was decreased in association with a shorter incubation time in a FXa inhibition assay and a thrombin inhibition-based activity test. However, the AT activity of R56C was comparable to that of WT when the incubation time was increased.

Conclusions

We concluded that the R56C mutant is responsible for type II HBS deficiency. We considered that the A459D and P112R mutants can be classified as belonging to the type I AT deficiency.