Thrombosis Research

Editorial Board

2010-08-01

Use of the Delphi method to establish guidelines for treatment of thrombosis

Jeremy Paikin, Mark Crowther — 2010-02-22

Pregnancy is a well-described risk factor for the development of venous thromboembolism (VTE). It is estimated that pregnancy confers up to a 4-fold increased risk of VTE when compared to age matched, non-pregnant patients . VTE is a relatively rare event in the pregnant patient, however, it remains the number one cause of pregnancy-related mortality in the developed world . Coincident thrombophilic risk factors further increase the risk of VTE . In the absence of large scale clinical trials, the optimal prophylaxis strategy for pregnant patients at high risk of thrombosis remains controversial. In this issue of the Journal, Chauleur et al. have used the Delphi method to develop a scoring system to help physicians: 1) predict thrombotic risk and 2) based on this risk, implement an appropriate prophylaxis strategy .

Acquired and Congenital Risk Factors associated with Cerebral Venous Sinus Thrombosis

Robert D. McBane, Alfonso Tafur, Waldemar E. Wysokinski — 2010-06-11

Abstract: The mechanistic paradigm underlying venous thrombosis at atypical locations stems from the observation that most events occur as a result of pathology of the organ system drained by the involved venous segment. Cerebral venous sinus thrombosis stands apart as an exception to this general rule. Although brain and sinus pathology are well established causes, these combined variables account for approximately one third of cases. The marked female preponderance and strong association with gender specific risk factors including hormonal manipulation, pregnancy and the puerperium are particularly notable. Factor V Leiden and prothrombin G20210A mutations and hyperhomocysteinemia represent important risk factors particularly when combined with acquired variables. The association with oral contraception use and the prothrombin G20210A gene mutation may offer insights into the anatomic predilection for cerebral venous sinus involvement as compared to venous thrombosis of the lower extremities. The intent of this review is to summarize the corporate literature of both acquired and congenital risk factors associated with cerebral venous sinus thrombosis in order to assist clinicians in their search for underlying mechanisms and to risk stratify patients for anticoagulation treatment duration and risk of recurrent thrombosis.

Use of the Delphi method to facilitate antithrombotics prescription during pregnancy

2010-02-22

Abstract: Introduction: Management of pregnant women at risk for venous thromboembolism (VTE) remains complex. Guidelines do not definitively fix optimal strategies due to limited trial data. Our objective was to build an easy-to-use tool allowing individualised, risk-adapted prophylaxis.Materials and Methods: A Delphi exercise was conducted to collect 19 French experts’ opinions on pregnancy-related VTE.Results: Experts with an active interest in clinical research and care of VTE and placental vascular complications were selected. The risk score was classified by an anonymous computer vote. A scoring system for VTE risk in pregnant women was developed, each score being associated with a specific treatment: graduated elastic compression stockings, aspirin, prophylactic Low Molecular Weight Heparin (LMWH: variable durations), or adjusted-dose of LMWH through pregnancy and postpartum.Conclusions: Our simple consensual scoring system offers an individual estimation of thrombosis risk during pregnancy together with its related therapeutic strategy, in accordance with most of the new international recommendations. The accuracy of our individual risk score-based therapeutic guidance is currently being prospectively evaluated in a multicenter trial (Clinicaltrials.gov registry no: NCT00745212).

Prophylactic therapy with enoxaparin in children with acute lymphoblastic leukemia and inherited thrombophilia during L-asparaginase treatment

2010-05-24

Abstract: Introduction: Thrombotic events (TE) are well documented in patients with acute lymphoblastic leukemia (ALL). They occur due to a combination of disease, host and treatment-related risk factors. Low molecular weight heparin (LMWH) has been found to be effective and safe in children with ALL during L-asparaginase treatment. At present, whether or not to give primary anticoagulant prophylaxis for TE during induction or reinduction courses to children with ALL is controversial. Our group investigated the use of LMWH as a prophylactic treatment for ALL children with a genetic prothrombotic predisposition.Methods: Eighty consecutive children with ALL treated between the years 1999 and 2008 were studied. Genetic analysis of factor V Leiden (G1691A) and prothrombin (G20210A) gene mutations were done at diagnosis. LMWH was given once daily subcutaneously at a dose of 1mg/kg, starting with the first dose of L-asparaginase (day 12 of induction, day 8 of consolidation) until one week after the last dose (day 40 of induction, day 25 of consolidation), to patients with inherited thrombophilia stemming from either factor V Leiden or prothrombin gene mutation.Results: Eighteen patients were found to have a genetic predisposition for TE, all of them received prophylactic LMWH. Six of the 80 (7.5%) patients developed thromboembolic events. Three of these six had a prothrombin (PT) gene mutation and received prophylactic LMWH. No TE event occurred in patients with factor V Leiden mutation receiving prophylactic LMWH.Conclusion: It is suggested that patients with ALL and PT gene mutation may have a higher risk of clotting complications in comparison to patients with factor V Leiden mutation. A randomized trial of LMWH should be performed to assess its safety and efficacy in preventing venous TE.

Seasonal variation in the incidence of superficial venous thrombophlebitis

2010-05-31

Abstract: Introduction: Previous studies have demonstrated an increased frequency and severity of symptoms due to varicose veins during summer. However there is no data on their complications, including superficial venous thrombophlebitis (SVT). The aim of this study was to test the hypothesis that SVT follows a seasonal pattern.Materials and Methods: During the two-year period between January 2007 and December 2008, inclusive, 123 patients with SVT were evaluated, including 60 females and 63 males. In 8 patients (6.5%) an additional and/or other predisposing factor was present. On presentation, SVT was complicated by thrombus extension to the proximal deep system in 5 cases (4.1%); above-knee SVT was present in 4 of these 5 cases.Results: SVT occurred more often during the months of May through October (monthly incidence of 7.3 cases) compared to remaining of the year (monthly incidence of 2.9 cases). SVT showed a peak in June and July with 33.3% of all SVTs occurring during these two months (monthly incidence of 10.25 cases). Using time-series statistics SVT occurrence showed a periodical seasonal pattern (p=0.003). Although a seasonal pattern was evident in all patient subgroups, this was significant only in males and patients with below-knee SVT.Conclusions: SVT showed a clear seasonal pattern of occurrence, with a significant rise during summer time. Although a possible explanation of this observation could be poor patient compliance and suboptimal usage of elastic stockings during the hot Mediterranean summer, further studies to investigate the cause, clinical significance and preventive methods of this complication are justified.

Haemostatic profile of healthy premature small for gestational age neonates

2010-06-10

Abstract: Background: The pathogenetic profile of premature Small for Gestational Age (SGA) neonates is strongly related to their haemostatic equilibrium, which is inadequately understood.Objective: To evaluate coagulation and fibrinolysis in premature SGA neonates before intervening with Vitamin K administration.Study design: We performed a comparison of coagulation, natural inhibitors and fibrinolysis between SGA and Appropriate for Gestational Age (AGA) infants born prematurely [gestational age (G.A.) <37weeks]. Study population consisted of 139 preterm newborns, 68 of whom were SGA (25 males and 43 females), while 71 were AGA (37 males and 34 females) that consisted the control group. Blood samples were obtained within 30minutes following birth and before the administration of vitamin K. Investigation included: PT, INR, APTT, fibrinogen, coagulation factors II, V, VII, VIII, IX, X, XI, XII, vWillebrand factor, protein C and free protein S, antithrombin (AT), APCR, tPA and PAI-1. The independent t-test and the Mann-Whitney U test were used to compare the differences between the values of haemostatic parameters.Results: Premature SGA infants presented significantly lower levels of fibrinogen (p<0.029) and higher levels of VIIIc factor, APCR, tPA and PAI-1 (p<0.041, 0.017, 0.021 and 0.019 respectively). The two groups had similar demographic characteristics (except from birth weight), without significant differences in the values of other haemostatic parameters.Conclusions: Despite the statistically significant differentiation in the levels of fibrinogen, VIIIc factor, APCR, tPA and PAI-1, the rest of haemostatic parameters have similar values between SGA and AGA preterms.

Magnetic resonance imaging and ultrasonography in diagnosis of pelvic vein thrombosis during pregnancy

2010-06-07

Abstract: Introduction: Pelvic deep vein thrombosis (DVT) is difficult to diagnose during pregnancy. In a two-center trial, we evaluated the agreement between ultrasonography and magnetic resonance imaging (MRI) in diagnosing the extent of DVT into the pelvic veins during pregnancy.Materials and methods: Pregnant women with proximal DVT were examined both with ultrasound and MRI as part of a study designed for treatment of DVT during pregnancy. Ultrasound was performed using color flow by specialist in vascular ultrasound with Doppler and compression techniques. The MRI sequences consisted of a 2D Time of Flight angiography with arterial flow suppression and maximum intensity projection reconstructions; a 3D, T1-w-prepared gradient echo sequence with fat saturation for thrombus imaging; a steady-state free precession sequence; and a Turbo-Spin-Echo. No contrast agent was used. Proportion of agreement (κ) for detection of DVT in individual veins was measured for different ipsilateral veins and inferior vena cava.Results: All 27 patients were imaged with both techniques at an average gestational age of 29 weeks (range 23-39). Three cases (11.5%) of DVT in the pelvic veins were missed on ultrasound but detected by MRI. The upper limit of the DVT was always depicted at a higher (20 cases, 65.4%) or the same level (seven cases, 34.6%) on MRI than on ultrasound. Agreement expressed as κ was 0.33 (95% CI 0.27-0.40) demonstrating only fair agreement. In one woman the thrombus had propagated into the inferior vena cava, shown only on MRI.Conclusion: Our study suggests that in pregnant women there is only fair agreement between ultrasound and MRI for determination of extent of DVT into pelvic veins, with MRI showing consistently more detailed depiction of extension. Our results indicate that MRI has an important role as a complementary technique in the diagnosis of DVT during pregnancy.

Leukocytosis, thrombosis and early mortality in cancer patients initiating chemotherapy

2010-06-07

Abstract: Background: Leukocytosis has been associated with thrombosis and mortality in cancer patients. We explored the association of leukocytosis with venous thromboembolism (VTE) and early mortality in cancer patients initiating chemotherapy.Methods: Data from a prospective, multicenter observational study of treatment-related complications in 4,405 ambulatory cancer patients initiating chemotherapy was used for this analysis. The association of leukocytosis, VTE and mortality during the course of chemotherapy was evaluated in univariate and multivariate analysis.Results: Ninety-three patients (2.1%) developed VTE and 134 (3%) died over a median follow up of 75days (range 0-384). Of 4391 patients with available baseline white blood cell (WBC) count, 561 (12.8%) had elevated pretreatment leukocyte counts, defined as WBC > 11×109 cells/L. VTE occurred in 25 of 561 patients (4.5%) with baseline leukocytosis compared to 68 of 3830 (1.8%) with WBC ≤ 11×109 cells/L (P<0.0001). Baseline leukocytosis was associated with VTE by multivariate analysis as well (HR 2.1, 95% confidence interval 1.3-3.4, p=0.003). Forty one patients (7.3%) with leukocytosis died compared to 92 (2.4%) with WBC ≤ 11×109 cells/L (P<0.0001). Baseline leukocytosis was associated with early mortality by multivariate analysis as well (HR 2.2, 95% confidence interval 1.5-3.3, p<0.0001). Mortality was greatest in patients with both leukocytosis and VTE. In multivariate analysis several factors were predictive of leukocytosis.Conclusions: Elevated WBC, particularly neutrophils, is strongly associated with increased risk of VTE and mortality in cancer patients receiving systemic chemotherapy. Further studies are needed to elicit the mechanisms involved.

The “normal” factor VIII concentration in plasma

2010-05-10

Abstract: Introduction: The quantitation of factor (F)VIII by activity-based assays is influenced by the method, procedure, the quality and properties of reagents used and concentrations of other plasma proteins, including von Willebrand factor (VWF).Objective: To compare FVIII concentrations measured by activity-based assays with those obtained by an immunoassay and to establish the influence of plasma dilution on the FVIII clotting activity (FVIIIc).Methods: The APTT, a chromogenic assay (Coatest) and two in-house immunoassays were used. Albumin-free recombinant FVIII was used as the calibrator in all assays.Results: For a group of 44 healthy individuals (HI), the mean value observed for FVIII antigen (FVIIIag; 1.22±0.56nM; S.D.) is substantially higher than that for FVIIIc (0.65±0.29nM) and the chromogenic assay (FVIIIch; 0.50±0.23nM). A positive correlation between FVIIIag and VWFag with R2=0.20 was observed. Since plasma VWF has an inhibitory effect on FVIIIc, we evaluated the influence of plasma dilutions on FVIIIc in HI (n=105). At a 4-fold dilution, estimates of FVIIIc by clotting assay were much lower than FVIIIag (0.77±0.31 vs. 1.14±0.48nM). At 10- and 25-fold dilutions, the estimated FVIIIc increased to 0.87±0.36 and 0.94±0.44nM, respectively.Conclusions: 1) In plasma, FVIIIag is higher than FVIIIc and FVIIIch; and 2) Real FVIII concentrations in plasma can be estimated by measuring FVIIIag.

Synergistic effect of a factor Xa inhibitor, TAK-442, and antiplatelet agents on whole blood coagulation and arterial thrombosis in rats

Noriko Konishi, Katsuhiko Hiroe, Masaki Kawamura — 2010-05-11

Abstract: Introduction: Activated platelets facilitate blood coagulation by providing factor V and a procoagulant surface for prothrombinase. Here, we investigated the potential synergy of a potent factor Xa/prothrombinase inhibitor, TAK-442, plus aspirin or clopidogrel in preventing arterial thrombosis and whole blood coagulation.Methods: Thrombus formation was initiated by FeCl3-induced rat carotid injury. Bleeding time was evaluated with the rat tail transection model. Whole blood coagulation was assessed by thromboelastographic examination (TEG) for which blood obtained from control, aspirin-, or clopidogrel-treated rats was transferred to a TEG analyzer containing, collagen or adenosine diphosphate (ADP), and TAK-442 or vehicle.Results: TAK-442 (3mg/kg, po), aspirin (100mg/kg, po) or clopidogrel (3mg/kg, po) alone had no significant effect on thrombus formation, whereas the combination of TAK-442 with aspirin and clopidogrel remarkably prolonged the time to thrombus formation without additional significant prolongation of bleeding time. TEG demonstrated that the onset of collagen-induced blood coagulation were slightly longer in aspirin-treated rats than control; however, when the blood from aspirin-treated rats was subsequently treated in vitro with 100nM TAK-442, the onset of clotting was significantly prolonged. In contrast, only marginal prolongation was observed with TAK-442 treatment of blood from control animals. The onset time of ADP-induced blood coagulation was slightly longer in clopidogrel-treated rats compared with control, and it was further extended by TAK-442 treatment.Conclusion: These results demonstrate that blood coagulation can be markedly delayed by the addition of TAK-442 to antiplatelets treatment which could contribute to synergistic antithrombotic efficacy in these settings.

A potential regulatory role for mRNA secondary structures within the prothrombin 3'UTR

Xingge Liu, Yong Jiang, J. Eric Russell — 2010-05-31

Abstract: The distal 3'UTR of prothrombin mRNA exhibits significant sequence heterogeneity reflecting an inexact 3′-cleavage/polyadenylation reaction. This same region encompasses a single-nucleotide polymorphism that enhances the normal post-transcriptional processing of nascent prothrombin transcripts. Both observations indicate the importance of 3'UTR structures to physiologically relevant properties of prothrombin mRNA. Using a HepG2-based model system, we mapped both the primary structures of reporter mRNAs containing the prothrombin 3'UTR, as well as the secondary structures of common, informative 3'UTR processing variants. A chromatographic method was subsequently employed to assess the effects of structural heterogeneities on the binding of candidate trans-acting regulatory factors. We observed that prothrombin 3'UTRs are constitutively polyadenylated at seven or more positions, and can fold into at least two distinct stem-loop conformations. These alternate structures expose/sequester a consensus binding site for hnRNP-I/PTB-1, a trans-acting factor with post-transcriptional regulatory properties. hnRNP-I/PTB-1 exhibits different affinities for the alternate 3'UTR secondary structures in vitro, predicting a corresponding regulatory role in vivo. These analyses demonstrate a critical link between the structure of the prothrombin 3'UTR and its normal function, providing a basis for further investigations into the molecular pathophysiology of naturally occurring polymorphisms within this region.

Microscopic clot fragment evidence of biochemo-mechanical degradation effects in thrombolysis

Franci Bajd, Jernej Vidmar, Aleš Blinc, Igor Serša — 2010-06-01

Abstract: Introduction: Although fibrinolytic treatment has been used for decades, the interactions between the biochemical mechanisms and the mechanical forces of the streaming blood remain incompletely understood. Analysis of the blood clot surface in vitro was employed to study the concomitant effect of blood plasma flow and recombinant tissue plasminogen activator (rt-PA) on the degradation of retracted, non-occlusive blood clots. Our hypothesis was that a faster tangential plasma flow removed larger fragments and resulted in faster overall thrombolysis.Materials and Methods: Retracted model blood clots were prepared in an optical microscopy chamber and connected to an artificial perfusion system with either no-flow, or plasma flow with a velocity of 3cm/s or 30cm/s with or without added rt-PA at 2µg/ml. The clot surface was dynamically imaged by an optical microscope for 30min with 15s intervals.Results: The clot fragments removed during rt-PA mediated thrombolysis ranged in size from that of a single red blood cell to large agglomerates composed of more than a thousand red blood cells bound together by partly degraded fibrin. The average and the largest discrete clot area change between images in adjacent time frames were significantly higher with the faster flow than with the slow flow (14,000μm2 and 160,000μm2 vs. 2200μm2 and 10,600μm2).Conclusions: On the micrometer scale, thrombolysis consists of sequential removal of clot fragments from the clot surface. With increasing tangential plasma flow velocity, the size of the clot fragments and the overall rate of thrombolysis increases.

Optimizing thrombelastography (TEG) assay conditions to monitor rFVIIa (NovoSeven®) therapy in haemophilia a patients

Dorthe Viuff, Søren Andersen, Brit B. Sørensen, Stefan Lethagen — 2010-06-10

Abstract: Introduction: There is no established laboratory method that can predict the most optimal dose of bypassing agents for treatment of haemophilia A. The objectives of the study was to develop an assay that can a) differentiate between the haemostatic capacity in blood from healthy individuals and severe and moderate haemophilia patients; b) show a dose-response correlation to rFVIIa addition; and c) show dose response differences of rFVIIa addition to plasma samples from non-inhibitor patients of different severity.Materials and Methods: Citrated whole blood from 25 haemophilia A patients was used in four thrombelastography (TEG) assays initiated with: 1) kaolin, 2) Tissue Factor (TF, Innovin 1:42,500), 3) TF 1:42,500+1.2nM tPA (tissue plasminogen activator) or 4) TF 1:200,000. rFVIIa was added to give a final concentration in the range of 0.02-4.8µg/ml.Results: The TEG assays showed large differences in clot formation demonstrated by prolonged clotting time (R-time), decreased maximum thrombus generation (MTG) between severe and moderate haemophilia A patients and between haemophilia patients and healthy males. The maximal amplitudes (MA) of the clot and resistance against fibrinolysis were only compromised when TF with tPA was added.Conclusion: In vitro addition of rFVIIa improved all TEG profiles significantly in a dose-dependent manner; but only the TEG assay containing kaolin could differentiate between the rFVIIa doses, showing that blood from severe patients need higher doses of rFVIIa to normalize the clot formation profile compared to blood from moderate patients. Kaolin seems to be the most useful TEG assay for monitoring rFVIIa treatment.

Prevalence and significance of anti-prothrombin (aPT) antibodies in patients with Lupus Anticoagulant (LA)

2010-06-09

Abstract: Objective: Anti-prothrombin (aPT) antibodies have been found in Lupus Anticoagulant (LA) positive patients. Their prevalence and relative contribution to thromboembolic risk in LA-positive patients is not well defined. The aim of this study was to determine their presence and association with thromboembolic events in a large series of patients with confirmed LA.Methods: Plasma from LA-positive patients was collected at Thrombosis Centers and sent to a reference central laboratory for confirmation. Positive plasma was tested using home-made ELISA for the presence of aPT and anti-β2GPI antibodies.Results: LA was confirmed in 231 patients. Sixty-one of 231 (26%, 95%CI 22-33) LA positive subjects were positive for IgG aPT and 62 (27%, 95% CI 21-33) were positive for IgM aPT antibodies. Clinical features of Antiphospholipid Syndrome (APS) were not associated with the presence of IgG aPT [43 APS in 61 (70%) positive and 109 APS in 170 (64%) negative IgG aPT subjects, p=ns] or IgM aPT. Rate of positivity of IgG and IgM aβ2GPI was significantly higher than that of IgG and IgM aPT. Clinical events accounting for APS occurred in 97 of 130 (75%) IgG aβ2GPI positive and in 55 of 101 (54%) IgG aβ2GPI negative patients (OR 2.4, 95% CI 1.4 to 4.3, p=0.002). No significant association with clinical events in patients positive for both IgG aPT and IgG aβ2GPI as compared to those positive for one or another test was found. When patients negative for both IgG aPT and IgG aβ2GPI (LA positive only) were compared with remaining patients, a significantly lower association with clinical events was found (OR=0.4, 95% CI: 0.2 to 0.7, p=0.004).Conclusions: As compared to IgG aβ2GPI, the prevalence of IgG aPT in patients with LA is significantly lower and not associated with the clinical features of APS.

Enhanced thrombin formation and fibrinolysis during acute Puumala hantavirus infection

2010-06-28

Abstract: Introduction: Nephropathia epidemica (NE) is a viral hemorrhagic fever with renal syndrome associated with thrombocytopenia and mild bleeding. We assessed activation of coagulation and fibrinolysis during the acute phase of NE.Materials and methods: 19 hospital-treated patients were involved. Plasma levels of D-dimer, prothrombin fragments 1+2 (F1+2), activated partial thromboplastin time (APTT), prothrombin time (PT%), thrombin time (TT), fibrinogen, antithrombin (AT), protein S free antigen (PS), protein C (PC) and complete blood count (CBC) were measured three times during the acute phase and once at 32-54days after the onset of fever (recovery phase). Laboratory abnormalities were evaluated by the disseminated intravascular coagulation (DIC) scoring advocated by the International Society of Thrombosis and Haemostasis (ISTH).Results: APTT was prolonged and D-dimer and F1+2 increased during the acute phase of NE. AT, PC and PS decreased, and TT was shortened, all implying increased thrombin generation. Acutely F1+2 was 3.4-fold and D-dimer even 24-fold higher compared with the recovery phase (median 726 vs 213pmol/l, and median 4.8 vs 0.2mg/l, respectively, p<0.001 for both). Platelet count correlated with AT, PC, and PS (r=0.73, r=0.81, and r=0.71, respectively, p<0.001 for all) as well as with fibrinogen (r=0.72, p<0.001). Only five patients fulfilled the ISTH diagnosis of DIC.Conclusions: During acute NE thrombocytopenia was associated with decreased natural anticoagulants, shortened thrombin time and enhanced fibrinolysis. Augmented thrombin formation and fibrinolysis characterize this hantavirus infection.

Factor XIII A subunit Val34Leu polymorphism in patients suffering atherothrombotic ischemic stroke

Amir H. Shemirani, Endre Pongrácz, Bálint Antalfi, Róza Ádány, László Muszbek — 2010-07-08

Abstract: Introduction: Factor XIII (FXIII) is a key regulator of fibrinolysis and clot firmness. Val34Leu polymorphism of its potentially active A subunit (FXIII-A) leads to faster activation of FXIII, influences clot structure and provides a moderate protection against coronary artery disease. The effect of FXIII A subunit (FXIII-A) Val34Leu polymorphism on the risk of ischemic stroke (IS) has been investigated in a few studies with contradictory results. In spite of their fundamental difference in pathogenesis and hemostatic pathomechanism, only four small studies investigated the effect of FXIII-A Val34Leu polymorphism on the risk of atherothrombotic IS (AIS) separately from cardioembolic IS. Gender specific effect of the polymorphism on the risk of AIS has not been explored. In the present study we investigated the effect of FXIII-A Val34Leu polymorphism on the risk of AIS on a large patient population.Materials and methods: A population control group of 1,146 randomly selected individuals, 496 patients surviving AIS and their age and sex-matched controls selected from the population control group were included in the study. FXIII-A Val34Leu genotype was determined on DNA samples, obtained from peripheral blood leukocytes, by fluorescence resonance energy transfer detection using melting curve analysis.Results: Neither sex nor age affected the distribution of FXIII-A Val34Leu genotypes in population control group. No association was revealed between the risk of AIS and FXIII-A Leu34 carriership and homozygous or heterozygous presentation of Leu34 allele in either gender.Conclusion: FXIII-A Val34Leu polymorphism fails to influence the risk of AIS.

Peri-procedural anticoagulation in patients undergoing ablation for atrial fibrillation

Sara R. Vazquez, Stacy A. Johnson, Matthew T. Rondina — 2010-02-22

Abstract: Radiofrequency catheter ablation is being used with increasing frequency as a strategy to manage atrial fibrillation. Patients undergoing this procedure are at increased short-term risk of thromboembolism for several days and up to 4weeks or longer after their ablation, and anticoagulation management surrounding the ablation procedure remains controversial. Although no conclusive recommendations can be made, published guidelines and data support therapeutic anticoagulation with warfarin for 3weeks prior and intravenous heparin during the ablation. Warfarin may either be continued through the ablation or stopped 2–5 days prior. If the latter approach is chosen, a pre-ablation bridging strategy of enoxaparin 1mg/kg twice daily is reasonable in selected patients unless the patient’s bleeding risk dictates using a lower dose regimen (0.5mg/kg twice daily) or avoiding bridging altogether. Fewer data are available for post-ablation management strategies, and current practice patterns are based largely on single-center experiences in smaller, non-randomized studies. For lower risk patients (CHADS2 0–1), either warfarin or aspirin may be utilized without bridging. In higher thromboembolic risk patients (CHADS2≥2), either enoxaparin (1mg/kg twice daily) or heparin may be started within the first 12–24h post-procedure. For patients with bleeding risk factors, enoxaparin may be subsequently reduced to 0.5mg/kg until the INR is therapeutic, although the efficacy of this lower dosing regimen has not been well studied. In accordance with national guidelines, warfarin should be continued post-ablation for a minimum of 2months and then indefinitely in patients with a CHADS2 score ≥ 2.

Association between factor V Leiden mutation and poor pregnancy outcomes among Palestinian women

Ayman S. Hussein, Hisham Darwish, Khaled Shelbayeh — 2010-06-02

Abstract: Pregnancy is a hypercoagulable state with increased tendency for thrombus formation, a condition that is increased when combined with acquired or inherited risk factors that lead to thrombophilia. Among the inherited risk factors is Factor V Leiden mutation, an autosomal dominant trait with reduced penetrance. The mutation seems to be associated with different poor pregnancy outcomes including recurrent miscarriages. In the present study, we performed a case-control study to investigate the association between the Leiden mutation and poor pregnancy outcome among the Palestinian population in the West bank region of Palestine. The study included 145 subjects with recurrent miscarriages and 205 matched control subjects with successful pregnancies who experienced normal delivery and no apparent complications. Leiden mutation was detected in 41 of the145 study subjects (28.2%), and in 24 of the 205 control subjects (11.7%). Subjects homozygous with the mutant allele were identified only among the test and not the control group. Data analysis indicates a significant association between the mutant allele and recurrent miscarriages (p-value<0.05). Furthermore, this association is significant between the mutant haplotype with miscarriages compared to control group showing time effect where there is no association for miscarriages before week 10. Results here also show strong association of factor V leiden polymorphism among primary aborters compared to secondary aborters or control groups. The odds ratio for the primary aborters was 75 and p<0.0001. In conclusion, these results provide evidence for a significant correlation between recurrent miscarriages and Factor V mutation in our population.

Antiplatelet effects of antidepressant treatment: A randomized comparison between escitalopram and nortriptyline

2010-05-31

Abstract: Introduction: Depressive disorders have been identified as independent risk factors for coronary heart disease. The present study (i) compared platelet function of depressed patients with that of healthy controls, (ii) analysed possible aggregability changes during 3months of treatment with antidepressants, and (iii) sought to assess different effects of escitalopram and nortriptyline on platelet aggregation.Methods: Blood samples of 91 major depressed patients and 91 healthy controls were analysed with whole blood aggregometry in a case-control setting. Depressed patients were randomized to two groups treated either with escitalopram (n=47) or nortriptyline (n=44). Platelet aggregation was studied on days 0, 1, 3, 7, 14, 21, 84 of continuing medication and was determined in response to adenosine diphosphate (ADP) and collagen.Results: Platelet aggregation induced by ADP was increased among depressive patients compared with that of healthy controls (26%, p=0.006). With antidepressant treatment, changes in platelet aggregation remained comparable in both groups at early time points (d1 to 21). In contrast, at day 84, patients with antidepressive response revealed significant differences in both medication groups: Patients receiving escitalopram showed a 23% decrease of ADP induced aggregation (p=0.03) and a 15% decrease of collagen induced aggregation (p=0.03). With nortriptyline the increase in impedance was reduced by 29% after ADP induction (p=0.046).Conclusion: Depressed patients have higher ex vivo platelet aggregation that may contribute to increased cardiovascular morbidity. After three months of antidepressant treatment with either escitalopram or nortriptyline, platelet aggregation was significantly reduced in antidepressant responders, irrespective of the antidepressant medication type.

Reducing agents induce thrombomodulin shedding in human endothelial cells

2010-06-07

Abstract: The level of thrombomodulin (TM) on cell surfaces reflects its biosynthesis, intracellular turnover, proteolytic cleavage, and release in soluble form (sTM). In the present study we examined the mechanisms mediating and regulating sTM release. Inducers of endothelial protein C receptor (EPCR) shedding, such as proinflammatory cytokines, phorbol ester, and ionomycin did not affect sTM release from human umbilical endothelial cells (HUVECs). In contrast, several natural and synthetic reducing compounds (i.e., glutathione, dihydrolipoic acid, homocysteine, N-acetyl-L-cysteine, dithiothreitol, and non-thiol cell-impermeable reductant, tris-(2-carboxyethyl)phosphine), but not oxidized glutathione or α-lipoic acid effectively up-regulated the release of sTM in endothelial cells. In addition, the direct activator of metalloproteases, 4-aminophenylmercuric acetate (APMA), was an effective inducer of TM shedding. Considerable inhibition of protein C activation was found with APMA, which is consistent with the effects of this agent on TM shedding. In addition to metalloproteases, serine proteases were shown by pharmacological inhibition studies to be involved in a similar degree in basal sTM release; however, serine proteases seem preferentially to be involved in thiol-induced TM proteolytic processing. From comparisons of non-thiol containing synthetic substrate with human recombinant TM it was demonstrated that disulfide bonds within TM are most likely modified by thiols making TM more susceptible to serine protease-mediated cleavage. In summary, the study shows that the extracellular redox state plays a crucial role in the regulation of TM shedding in HUVECs thereby offering new strategies to interfere with diminished activation of protein C during inflammatory diseases.

Contribution of neutrophil elastase to the lysis of obliterative thrombi in the context of their platelet and fibrin content

2010-06-07

Abstract: Leukocytes invade newly formed thrombi through interactions with platelets and fibrin and later contribute to the removal of fibrin deposits mainly through the action of neutrophil elastase. The present study attempts to express in quantitative terms the impact of neutrophils on the lytic processes in obliterative thrombi based on the local presence of elastase-specific fibrin degradation products (NE-FDP) in relation to the leukocyte, platelet and fibrin content of thrombi. Immunofluorescent detection of fibrin, NE-FDP and platelet antigens was performed in sections of thrombi from 28 patients subjected to thrombectomy in combination with DNA-staining for identification of nucleated cells. The digitalized fluorescent microscopic images were decomposed according to the color channel of each thrombus constituent. The integrated intensity values for all thrombus constituents were statistically evaluated with correlation, hierarchical agglomerative clustering , Hotelling'sT2 and F-statistics. Association between NE-FDP and leukocyte content of thrombi is evidenced by a significant Pearson correlation coefficient of 0.71 (p=0.00002). Cluster analysis reveals two classes of thrombi according to NE-FDP, leukocyte and platelet content and also two according to NE-FDP, leukocyte and fibrin content. When NE-FDP, fibrin and platelet content is normalized to the leukocyte count in the same thrombus, clusters with platelet-related thrombolytic resistance (inversely related NE-FDP and platelet content) and advanced cell-dependent thrombolysis (inversely related NE-FDP and fibrin content) are identified. These distinct patterns of thrombus constituents are snapshots of characteristic stages in the cell-dependent thrombolysis, which indicate a clot-stabilizing role for platelets in this process similar to their impact on the plasmin-dependent lysis.

Standardized antigen preparation to achieve comparability of anti-β2-glycoprotein I assays

2010-06-18

Abstract: Introduction: The Sydney classification for diagnosis of the antiphospholipid syndrome (APS) first introduced the determination of anti-β2-glycoprotein I (anti-β2-GPI)-antibodies in serum as laboratory criteria. In this context, widely differing results of anti-β2-GPI assays are a concerning issue. Considerable efforts have been made to optimize ELISAs, however little attention was hitherto spent to the antigen preparation. We evaluated the influence of different β2-GPI preparations on the ability to separate ill and healthy patients and on the comparability of anti-β2-GPI-assays.Materials and Methods: Microplates were coated with various β2-GPI preparations and anti-β2-GPI IgG- and IgM-ELISAs were performed for 21 APS patients and 21 controls using the monoclonal calibrators HCAL and EY2C9. Subsequently, by use of a surface plasmon resonance (SPR) biosensor, affinity constants for the HCAL- and EY2C9-interaction with each β2-GPI preparation were determined and antigen binding of sera of APS patients and controls was studied.Results: All ß2-GPI preparations showed good discrimination ability ill vs. healthy but poor inter-assay comparability in the ELISAs. Affinity constants for HCAL and EY2C9 were independent of the β2-GPI variant (KA 0.105 – 0.200 and 0.449 – 1.04×1010M-1; KD 50.0 – 95.5 and 9.61 – 22.3×10-11M, respectively). In the biosensor, reactivity to the different β2-GPIs was negligible for the controls and varied considerably for patients.Conclusion: Inter-assay comparability of anti-β2-GPI ELISAs is highly dependent upon the β2-GPI preparation. Only agreement on one common β2-GPI preparation will improve the requested inter-assay comparability.

EMPoWarMENT: Edmonton Pediatric Warfarin Self-Management Pilot Study in Children with Primarily Cardiac Disease

2010-06-28

Abstract: Increasing numbers of children require warfarin thromboprophylaxis. Home INR testing by the patient (PST) has revolutionized warfarin management. However, the family/patient must contact the health team for guidance for warfarin dosing. Patient self management(PSM) prepares a patient performing PST to take an active role in warfarin dosing. Adult studies demonstrate that PSM is safe and effective with improved adherence and treatment satisfaction quality of life (QOL).Objective: To estimate the safety and efficacy in children performing PSM or PST, to evaluate warfarin dose decision making in PSM, and warfarin related QOL.Methods: Warfarinized children performing PST for >3m were randomized to PST or PSM. The PSM group underwent warfarin management education and assumed independent warfarin management. INRs were collected for a year prior to and for 1year of study to determine TTR and warfarin decision making. QOL was assessed through inventory completion and interviews.Results: 28 children were randomized and followed for 12months. TTR was (83.9% pre/ post), and 77.7% pre to 83.0% post for PST and PSM (p=0.312). Appropriate warfarin decision making was 90% with no major bleeding episodes and no thromboembolic events. PSM was preferred by families. Conclusions: PSM for children may be a safe and effective management strategy for warfarinized children. Clinical studies with larger sample size are required.

Development and preliminary evaluation of the KIDCLOT PAC QL©: A new health-related quality of life measure for pediatric long-term anticoagulation therapy

A.A.K. Bruce, M.E. Bauman, K. Black, A. Newton, L. Legge, M.P. Massicotte — 2010-06-28

Abstract: Long term anticoagulation (LTA) is hypothesized to induce treatment dissatisfaction influence quality of life (QOL). QOL is measured by a tool developed specific to the patient condition. Pediatric QOL inventory for children on LTA should assess constructs salient for this population. Identification and evaluation of QOL constructs, critical to improve care, and is accepted as the "gold-standard" measurement for patient-centered outcomes in clinical research.Objectives: To develop and preliminarily validate a pediatric QOL inventory for children/families receiving LTA. Secondary objective was to determine how anticoagulation disrupts children's life.Methods: Stage 1: Item/theme generation through focus groups and existing inventories, Stage 2: Item reduction, inventory generation and content validity. Stage 3: Inventory refinement, implementation and reliability testing. Responses were evaluated for variability, internal consistency, and scale structure. Item reduction was based on response rate, item variability, and clinical utility.Results: Two inventories, KIDCLOT-PAC-Child –Tween QL© and KIDCLOT-PAC Parent-proxy-QL © were developed. Content and face validity was assessed by experts, parents, and patients. Internal consistency determined by Cronbach's α was good for parent-proxy(0.82) and child(0.89). Pearson correlation was acceptable with >0.5 for test-retest reliability (parent inventory).Conclusions: KIDCLOT-PAC-QL© is the first preliminarily validated inventory to assess QOL in anticoagulated children. The inventory identifies barriers in care and areas for improvement in order to modify care to provide the "best" management (improved QOL associated with safety and efficacy) for children requiring LTA.

Coagulation factor XIII activation peptide and subunit levels in patients with acute ischaemic stroke: A pilot study

2010-06-28

Abstract: Introduction: We have recently shown that FXIII activation peptide (AP-FXIII) can be measured in plasma. The objective of this pilot study was to investigate for the first time if AP-FXIII can be detected in plasma from patients with acute ischaemic stroke.Materials and methods: We included 66 patients with acute ischaemic stroke admitted between 1 and 7hours after the onset of clinical symptoms. We collected plasma samples upon admission and on the two following days and measured AP-FXIII and subunit levels by ELISA. Clinical stroke severity was assessed by NIHSS stroke score.Results: AP-FXIII could be detected in 34 patients upon admission (range 0.2-26.3ng/ml), on day 1 in 15 patients (0.2-10.4ng/ml), and on day 2 in 11 patients (0.1-15.1ng/ml. AP-FXIII was higher in patients with severe stroke. Lower AP-FXIII levels upon admission were associated with clinical improvement. FXIII-A and FXIII-B subunit levels decreased significantly from day 0 to day 1.Conclusions: For the first time, we detected AP-FXIII in patients upon an acute thrombotic event. The decrease in FXIII subunit levels during acute ischaemic stroke is evidence for ongoing coagulation activity and FXIII consumption. Our results suggest that FXIII activation and concomitant AP-FXIII release might be associated with an unfavourable short-term clinical outcome. Larger studies are needed to further investigate whether AP-FXIII might serve as a diagnostic and/or prognostic marker for acute thrombotic diseases.

Plasma activity of individual coagulation factors, hemodilution and blood loss after cardiac surgery: A prospective observational study

2010-06-28

Abstract: Background: Hemodilution and consumption of coagulation factors during cardiopulmonary bypass has been suggested to contribute to bleeding complications after cardiac surgery. The aim was to describe the activity of individual coagulation factors after CABG in relation to hemodilution and postoperative bleeding.Materials and Methods: Plasma concentrations of fibrinogen and plasma activity of FII, FV, FVII, FVIII, FIX, FX, FXI and FXIII adjusted for hemodilution were analysed in 57 CABG patients before, and 2h and 24h after surgery. Postoperative bleeding was registered and correlations to coagulation factor activity were calculated.Results: Adjusted plasma concentration of fibrinogen (-14±6%), and plasma activity of FII (-9±6%), FV (-13±8%), FX (-13±7%) and FXIII (-9±14%) were reduced two hours after surgery compared to baseline (all p<0.001). FVII (+3±12%, p=0.34) and FXI (+1±19%, p=0.50) were unchanged, while FVIII (+23±44%, p=0.006) and FIX (+23±17%, p<0.001) increased. Twenty-four hours after surgery fibrinogen (+45±27%), FVIII (+93±66%) and FIX (+33±26%) were all increased (all p<0.001), while FVII (-37±14%, p<0.001), FXI (-4±18%, p=0.02) and FXIII (-6±15%, p=0.004) were decreased.Median postoperative blood loss was 380ml/12h. There were significant inverse correlations between postoperative blood loss and fibrinogen concentration 2h after surgery (r=-0.33, p=0.019) and between postoperative blood loss and pre- and postoperative FXIII activity (r=-0.34, p=0.009 and r=-0.41, p=0.003, respectively), but not between blood loss and any of the other factors.Conclusions: There is a marked dissociation in plasma activity of individual coagulation factors after CABG. Plasma concentration of fibrinogen and factor XIII activity correlates inversely to postoperative blood loss after CABG.

VKORC1 and CYP2C9 genetic polymorphisms in hepatic or portal vein thrombosis

2010-05-03

Hepatic or portal vein thrombosis is a rare disease, usually caused by multiple concurrent factors, including acquired and inherited thrombophilias . Long term anticoagulation therapy with vitamin K antagonists (VKA) is currently recommended in this clinical setting with a target international normalized ratio (INR) comprised between 2 and 3 . However, over anticoagulation has proven to be one of the leading causes of bleeding episodes that are the most serious complication in these patients, major haemorrhages often affecting the gastrointestinal tract because of oesophageal varices. Polymorphisms of the VKORC1 (vitamin K epoxide reductase complex, subunit 1) gene and the CYP2C9 (cytochrome P450, family 2, subfamily C, polypeptide 9) gene have been shown to affect INR responses and the bleeding risk associated with VKA prophylaxis . VKORC1 is part of a multiprotein complex that recycles vitamin K 2,3-epoxide to vitamin K hydroquinone, a cofactor that is essential for the post-translational gamma-carboxylation of several blood coagulation factors including factors II (prothrombin), VII, IX, and X . CYP2C9 is responsible for the metabolic clearance of the more pharmacologically potent S-enantiomer of warfarin (a VKA coumarin derivative) . VKORC1 and CYP2C9 genotypes, age, and height were estimated to account for nearly 55% of the variability in warfarin daily dose requirements . In addition, it remains controversial whether VKORC1 could influence the risk of venous thromboembolism and/or arterial vascular disease . The putative association could rely on VKORC1-dependent effects on the vitamin K-dependent proteins such as clotting proteins, Matrix Gla protein (MGP) and osteocalcin. In the present retrospective study, we examined the distribution of the minor VKORC1 and CYP2C9 alleles in a population of patients with hepatic or portal vein thrombosis. The polymorphisms influence on the variability of response to VKA after one month treatment initiation was tested in the patients under long term oral anticoagulants.

The influence of citrate concentrations on PFA-100 closure times, platelet hyper-reactivity and aspirin monitoring

Paul Harrison — 2010-02-22

The recent published article by Nazarian et al measured shear dependent platelet reactivity using the PFA-100 device (Siemens Diagnostics, Marburg, Germany) within 288 patients on chronic aspirin therapy scheduled for Coronary Artery Bypass Grafting (CABG). The majority (N = 285 or 99%) of the patients were non-responsive to arachidonic acid aggregometry confirming that these individuals were indeed all responsive to their aspirin therapy. In contrast platelet hyper-reactivity as measured by normal closure times (CT) within the aspirin sensitive PFA-100 Collagen/Epinephrine (CEPI) cartridge were frequent especially in blood samples anticoagulated with 3.2% citrate compared to 3.8% (38.4% versus 13.5%, p<0.0001). Shortened CT's (below 71 seconds) were also frequently obtained in the aspirin insensitive Collagen/ADP (CADP) cartridge and again were more apparent in 3.2% citrated blood samples compared to 3.8% citrate (31.7% versus 17.8%, p<0.0001). The results are largely in agreement with previous literature, and the authors provide additional evidence that PFA-100 results are affected more by additional variables (e.g. VWF, Haematocrit, race and warfarin) within 3.2% than in 3.8% citrate anticoagulated blood (i.e. VWF only). The authors therefore correctly question the specificity of the CEPI cartridge for assessing aspirin resistance and it is clear that from this and other studies that the PFA-100 cannot be used to determine biochemical aspirin resistance that is specific to cyclooxygenase-1 (COX-1) activity. However, recent meta-analyses do show the potential utility of the test for detecting on aspirin treatment platelet hyper-reactivity and its significant association with major adverse clinical events (MACE). A higher prevalence of shortened closure times within 3.2% citrate compared to 3.8% citrate anticoagulated blood was also confirmed in the meta-analysis of 6500 aspirin treated patients tested on the PFA-100 . It is therefore very clear that aspirin resistance cannot be measured using the PFA-100 test and this definition is totally inappropriate using this methodology as the CEPI test result can often be largely independent of COX-1 inhibition. The potential clinical utility of detecting platelet hyper-reactivity in the PFA-100 deserves much further research as a relatively simple but physiological platelet test could be useful for detecting and stratifying patients at high risk of thrombosis.

Lupus Anticoagulant-Hypoprothrombinemia syndrome (HLAS): Report of one case in a familial infectious context

A. Appert-Flory, F. Fischer, J. Amiral, F. Monpoux — 2010-02-22

A previously healthy 2years old girl was admitted in the paediatric emergency room of our hospital for a gastrointestinal bleeding suspicion. There was no bleeding disorder in the family or patient history. Physical examination showed few bruises and the bleeding appeared to be a swallowed epistaxis. The patient suffered from mild upper respiratory tract infection probably in part responsible for the epistaxis.

Argatroban and renal replacement therapy in a morbidly obese patient with heparin-induced thrombocytopenia: A case report

2010-02-22

Continuous veno-venous hemofiltration (CVVH) is widely used for renal replacement therapy (RRT) in intensive care units (ICUs). However, because patients undergoing CVVH often receive unfractionated heparin (UFH), there is a risk of Heparin-Induced Thrombocytopenia (HIT) and also of repeated hemofilter thrombosis . Administration of a reversible direct thrombin inhibitor, such as argatroban or lepirudin, or of the heparinoid, danaparoid, is an alternative anti-coagulation strategy for RRT in patients with HIT. However, information on the use of argatroban in obese patients is relatively scarce. We report the case of a morbidly obese patient who received dose-adjusted argatroban for RRT with monitoring of anti-IIa activity.

Pediatric warfarin practice and pharmacogenetic testing

Courtney D. Thornburg, Emma Jones, Lisa Bomgaars, Brian F. Gage — 2010-02-22

Polymorphisms in cytochrome P450 (CYP) 2C9 and vitamin K epoxide reductase (VKORC1) are associated with warfarin dose . Warfarin dosing algorithms that incorporate pharmacogenetic (PGx) testing have been developed for adults and are being testing in clinical trials . In 2007, the FDA updated warfarin prescribing information, recommending PGx testing for CYP2C9 and VKORC1, but questions remain regarding the clinical utility of the PGx algorithms and how they will be integrated into clinical care, especially pediatric practice.

Relationship between platelet reactivity inhibition and non-CABG related major bleeding in patients undergoing percutaneous coronary intervention

2010-02-22

Dual anti-platelet regimen has dramatically decreased the rate of thrombotic events including stent thrombosis in patients undergoing percutaneous coronary intervention (PCI) . However following the report of a large inter-individual variability in response to clopidogrel loading dose (LD), several studies have demonstrated a strong relationship between the level of platelet reactivity inhibition (PRI) achieved and the occurrence of thrombotic events following PCI . The TRITON TIMI 38 and PRINCIPLE-TIMI 44 trials have demonstrated that achieving higher levels of PR inhibition using a more potent thienopyridine, prasugrel, compared to clopidogrel was associated with a reduction in the rate of major adverse cardiovascular events (MACE) . However in the TRITON TIMI 38 trial, patients randomized to receive prasugrel were more likely to suffer from TIMI major bleedings. Together, these findings have suggested a link between platelet reactivity inhibition and major bleedings . We therefore aimed to investigate the relationship between PRI following clopidogrel LD and in-hospital non-CABG related TIMI major bleedings.

The VKORC1 promoter is occupied by c-Myc transcription factor in HepG2 cells

Anna Pfister, Abdimajid Osman — 2010-02-22

Polymorphisms in the gene encoding vitamin K epoxide reductase (VKORC1) are associated with warfarin dose requirement . Particularly, the promoter SNP -1639C>T (rs9923231) is regarded as an important genetic marker and is associated with low warfarin dose requirement . The minor T-allele of -1639C>T SNP creates an E-box motif, CACCTG, expected to be a potential binding site for nuclear transcription factors. The VKORC1 gene is expressed in many cell types in the body and is not uniquely expressed in the liver. Thus, other cells than liver cells could be used to study VKORC1 expression. The human hepatocarcinoma cell line, HepG2, shows many liver-specific functions and is frequently used for VKORC1 transcriptional activity studies . However, HepG2 cells also display several abnormal cellular activities. For instance, HepG2 contains elevated levels of c-Myc and other oncogenic proteins. The Myc protein is a transcription factor that binds preferably to CACGTG E-box elements, although recent studies have revealed that c-Myc also binds to a number of other non-palindromic E-box elements . Interestingly, c-Myc was found to bind to CACCTG E-box at DHFR gene promoter , which is exactly identical to the element at VKORC1 -1639. We therefore wondered whether the over-expressed Myc protein in HepG2 interacts with VKORC1 promoter E-box and may be part of a transcription factor assembly at the promoter.

Automated APTT cycle for the rapid identification of plasma prekallikrein deficiency

2010-03-08

Very prolonged activated partial thromboplastin time (APTT) is a challenging issue for coagulation laboratories and warrants further investigation, once preanalytical or analytical causes have been excluded. Abnormal APTT may be due to anticoagulation therapy, primarily heparin, which can be present in the specimen as a therapeutic or contaminant agent, or it may be due to a coagulation factor deficiency, either congenital or acquired (e.g., presence of specific inhibitors directed against single coagulation factors or non-specific inhibitors, such as Lupus Anticoagulants). When haemophilia (A, B or C) and lupus anticoagulants (LAs) have been ruled out, abnormalities in the so-called contact system, which includes plasma prekallikrein, factor XII (FXII) and high molecular weight kininogen (HK), should be investigated. Prekallikrein, also known as Fletcher Factor, is converted to its active form (kallikrein) by FXII that can autoactivate on negatively charged artificial surfaces or in the presence of various biological substances . Plasma kallikrein in turn further activates FXII with consequent amplification of the process. Prekallikrein may also be activated by the serine protease prolylcarboxypeptidase and by heat shock protein 90 in a FXII-independent way. Deficiency of prekallikrein, which is not generally associated with bleeding tendency and is inherited as an autosomal recessive trait , is characterised by severely prolonged APTT that progressively shorten and even normalise when the preincubation time of plasma with the reagent containing a contact activator is extended to at least 10-20minutes . This behaviour is due to a bypass mechanism that allows that sufficient activated FXII is generated to further activate the coagulation factor XI . Shortening of prolonged APTT with extended incubation time of plasma with a contact activator before recalcification was described in LA samples but, in this case, did not achieve normalisation . APTT assays with extended incubation times are not commonly available on automated coagulometers; furthermore, the manual tilt-tube method is not feasible in all laboratories.

Clinical biological remission induced by rituximab in acute refractory chronic relapsing TTP

2010-03-02

Thrombotic thrombocytopenic purpura (TTP) is a rare but often fatal disorder characterized by thrombocytopenia, microangiopathic hemolytic anemia and microvascular thrombosis. These symptoms relate to the presence of von Willebrand factor (vWF)-rich platelet thrombi secondary to a congenital or acquired deficiency of ADAMTS13, a vWF-cleaving protease . Acquired forms are usually associated with the presence of anti-ADAMTS13 IgG that in most cases neutralize the enzyme's activity , though non-neutralizing antibodies have also been reported . Prognosis of TTP has been dramatically improved by prompt therapeutic plasma exchange (PE) . However, some acute severe forms are refractory to this treatment and 20-50% of patients develop a chronic, relapsing form needing further alternative treatments. In the last few years rituximab, a humanized anti-CD20 monoclonal antibody, has been proposed both as rescue therapy in acute forms of TTP refractory to PE and prophylactic treatment in relapsing patients who maintain a high anti-ADAMTS13 inhibiting antibody titre even when in clinical remission . In this retrospective report, we describe the clinical course of four idiopathic TTP patients treated with rituximab. Three patients were treated during their first PE-refractory episode, while the fourth was prophylactically treated during remission after his first relapse. ADAMTS13 activity and anti-ADAMTS13 IgG autoantibodies were assessed before and during rituximab treatment.

Autopsy case of antithrombin producing in hepatocellular carcinoma

Yasuo Ochiai, Toshiaki Kunimura, Toshio Morohoshi — 2010-03-29

Antithrombin (AT) is synthesized in the liver and is an important inhibitor of blood coagulation. Congenital AT deficiency and abnormal AT are well known causes of thrombosis. Low levels of AT are found in patients with nephritic syndrome , disseminated intravascular coagulation, liver cirrhosis, and hepatocellular carcinoma . Thus, an abnormally high increased level of AT in hepatic diseases should be extremely rare. Recently, we had a chance to examine an autopsy case of primary hepatocellular carcinoma with an abnormally high increased level of AT. We report here the results.

Severe arterial thrombosis in a family with type III protein S deficiency caused by a frameshift mutation in the PROS1 gene

Torben Bjerregaard Larsen, Klaus Brusgaard, Mads Nybo — 2010-04-20

Arterial thrombosis is commonly associated with atherosclerosis, smoking, hypertension, and diabetes, but the role of thrombophilia in determining the risk of arterial thrombotic events is less well defined . Hereditary protein S (PS) deficiency has been associated with myocardial infarction (MI), and an increased tendency of arterial thrombosis due to PS deficiency has been suggested . However, only few reports have described hereditary PS deficiency associated with recurrent MI . We here report a frameshift mutation in the PS gene (PROS1) associated with premature and fatal arterial thrombosis in a 40year old man followed by an investigation of several family members in order to elucidate the nature of PS deficiency.

Coexisting dysfibrinogenemia (γArg275His) and FV Leiden associated with thrombosis (Fibrinogen Crete)

2010-05-24

Dysfibrinogenemia is a rare congenital disorder characterised by mutations in either of the three fibrinogen genes (FGA, FGB, FGG) that encode for the three polypeptide chains (Aα,Bβ,γ) of the fibrinogen molecule. It is inherited as a dominant trait and most patients are heterozygous for missense mutations which allow secretion of the mutant fibrinogen in the circulation. Many dysfibrinogenemias have been characterized at the functional level, causative mutations have been found to have deleterious effects on fibrin polymerization, fibrin crosslinking by FXIIIa as well as fibrinolysis. Most carriers of this disorder are asymptomatic, while a minority may present with either thrombotic or bleeding complications. Development of thrombosis in patients with dysfibrinogenemia is usually associated with defects that cause abnormal fibrinopeptide release, defective polymerization, impaired interaction with platelets, defective fibrinolysis and abnormal calcium binding . Since most carriers are asymptomatic, it can be postulated that the presence of other inherited predisposing factors might trigger the development of thrombotic or bleeding complications in dysfibrinogenemia patients. More than 400 cases of dysfibrinogenemia have been reported so far, however there are only limited data on the coexistence of prothrombotic mutations in these patients and on the effect of double carriership on the risk for thrombosis. Reports addressing the involvement of additional predisposing genetic defects are interesting and strengthen the hypothesis that coexistence of multiple risk factors may trigger development of thrombosis among the rare dysfibrinogenemia panel.

Prevalence of Factor IX-R338L (Factor IX Padua) in a cohort of patients with venous thromboembolism and mild elevation of factor IX levels

2010-06-03

We read with interest the data presented by Simioni et al. demonstrating for the first time that a point mutation in the factor IX (FIX) gene (R338L) can be the cause of elevated FIX activity in patients with venous thromboembolism (VTE). In their article, Simioni and coworkers elegantly demonstrated that R338L is a gain-of-function mutation in FIX gene (which they called FIX Padua) that results in marked elevation of FIX activity. In their report, a 23year-old male with a history of spontaneous VTE and with the R338L mutation, presented FIX levels about eight times normal despite a normal FIX protein in plasma. The authors did not find any case of the point mutation 200 healthy individuals from the same geographic region, nor in 200 patients with documented VTE and normal FIX activity levels.