[New oral anticoagulants - influence on coagulation tests].

The new oral anticoagulants (NOACs) represent alternative antithrombotic agents for prophylaxis and therapy of thromboembolic diseases. They act either by inhibition of the clotting factor Xa or IIa (thrombin). As a consequence, they influence several coagulation assays (for example prothrombin time, activated partial thromboplastin time). Because of the short half-life of these new agents, these changes show great variations in the course of 24 hours. Furthermore, there are significant differences of laboratory results depending on the used reagents. We explain the influence of apixaban, rivaroxaban (factor Xa inhibitors) and dabigatran (thrombin inhibitor) on the most commonly used coagulation assays. Besides we show that this influence depends on the way of action of the drug as well as on the principle of the coagulation assay. Being aware of this relationships helps to interpret the results of coagulation assays under influence of NOACs correctly.

Reduced serum chemerin in patients with more severe liver cirrhosis

Chemerin is a well-established modulator of immune cell function and its serum levels are induced in inflammatory diseases. Liver cirrhosis is associated with inflammation which is aggravated by portal hypertension. The objective of this study was to evaluate whether chemerin is induced in patients with more severe liver cirrhosis and portal hypertension. Chemerin has been measured by ELISA in the portal venous serum (PVS), systemic venous serum (SVS) and hepatic venous serum (HVS) of 45 patients with liver cirrhosis. Chemerin is higher in HVS compared to PVS in accordance with our recently published finding. SVS, HVS and PVS chemerin decline in patients with more advanced liver injury defined by the CHILD-PUGH score. Hepatic chemerin has been determined in a small cohort and is similarly expressed in normal and cirrhotic liver. MELD score and serum markers of liver and kidney function do not correlate with chemerin. There is a positive correlation of chemerin in all compartments with Quick prothrombin time and of SVS chemerin with systolic blood pressure. PVS chemerin is induced in patients with modest/massive ascites but this does not translate into higher HVS and SVS levels. Chemerin is not associated with variceal size. Reduction of portal pressure by transjugular intrahepatic portosystemic shunt does not affect chemerin levels. These data show that low chemerin in patients with more severe liver cirrhosis is associated with reduced Quick prothrombin time.

Ancylostoma caninum anticoagulant peptide: a hookworm-derived inhibitor of human coagulation factor Xa.

Human hookworm infection is a major cause of gastrointestinal blood loss and iron deficiency anemia, affecting up to one billion people in the developing world. These soil-transmitted helminths cause blood loss during attachment to the intestinal mucosa by lacerating capillaries and ingesting extravasated blood. We have isolated the major anticoagulant used by adult worms to facilitate feeding and exacerbate intestinal blood loss. This 8.7-kDa peptide, named the Ancylostoma caninum anticoagulant peptide (AcAP), was purified by using a combination of ion-exchange chromatography, gel-filtration chromatography, and reverse-phase HPLC. N-terminal sequencing of AcAP reveals no homology to any previously identified anticoagulant or protease inhibitor. Single-stage chromogenic assays reveal that AcAP is a highly potent and specific inhibitor of human coagulation, with an intrinsic K*i for the inhibition of free factor Xa of 323.5 pM. In plasma-based clotting time assays, AcAP was more effective at prolonging the prothrombin time than both recombinant hirudin and tick anticoagulant peptide. These data suggest that AcAP, a specific inhibitor of factor Xa, is one of the most potent naturally occurring anticoagulants described to date.

Validation and refinement of survival models for liver retransplantation

Orthotopic liver retransplantation (re-OLT) is highly controversial. The objectives of this study were to determine the validity of a recently developed United Network for Organ Sharing (UNOS) multivariate model using an independent cohort of patients undergoing re-OLT outside the United States, to determine whether incorporation of other variables that were incomplete in the UNOS registry would provide additional prognostic information, to develop new models combining data sets from both cohorts, and to evaluate the validity of the model for end-stage liver disease (MELD) in patients undergoing re-OLT. Two hundred eighty-one adult patients undergoing re-OLT (between 1986 and 1999) at 6 foreign transplant centers comprised the validation cohort. We found good agreement between actual survival and predicted survival in the validation cohort; 1-year patient survival rates in the low-, intermediate-, and high-risk groups (as assigned by the original UNOS model) were 72%, 68%, and 36%, respectively (P < .0001). In the patients for whom the international normalized ratio (INR) of prothrombin time was available, MELD correlated with outcome following re-OLT; the median MELD scores for patients surviving at least 90 days compared with those dying within 90 days were 20.75 versus 25.9, respectively (P = .004). Utilizing both patient cohorts (n = 979), a new model, based on recipient age, total serum bilirubin, creatinine, and interval to re-OLT, was constructed (whole model χ(2) = 105, P < .0001). Using the c-statistic with 30-day, 90-day, 1-year, and 3-year mortality as the end points, the area under the receiver operating characteristic (ROC) curves for 4 different models were compared. In conclusion, prospective validation and use of these models as adjuncts to clinical decision making in the management of patients being considered for re-OLT are warranted.

Vitamin E and alpha-lipoic acid supplementation increase bleeding tendency via an intrinsic coagulation pathway

Vitamin E and a-lipoic acid are potent nutritional antioxidants, and when used together, their antioxidant capabilities are improved as a-lipoic acid recycles vitamin E. Supplementation of vitamin E has been shown to prolong platelet aggregation but the effects of vitamin E and alpha-lipoic acid supplementation on bleeding tendency have yet to be reported. Young, male rats consumed either control diet (n=5) or vitamin E and a-lipoic acid-supplemented diet (n=5) for 14 weeks. Activated partial thromboplastin time (APTT) and prothrombin time (PT) were measured as markers of intrinsic and extrinsic coagulation pathways respectively in addition to lipid peroxidation (malondialdehyde). Supplementation significantly prolonged APTT (23.8 +/- 1.5 vs 31.4 +/- 1.2s, p < 0.05) compared to the con-trol diet; however, there was no significant difference in PT (27.8 +/- 1.5 vs 26.6 +/- 0.9s, p > 0.05). While vitamin E was increased (p < 0.05), there was no significant difference in plasma levels of malondialdehyde (p > 0.05). Dietary supplementation of vitamin E and alpha-lipoic acid increases bleeding tendency via inhibition of the intrinsic coagulation pathway with no change in markers of lipid peroxidation. Such supplementation could benefit patients with cardiovascular disease who exhibit elevated levels of coagulation and oxidative stress.

Otimização da extração de polissacarídeos sulfatados da alga vermelha Gracilaria birdiae e análise da atividade anticoagulante e antioxidante

The sulfated polysaccharides (SP) from the edible red seaweed Gracilaria birdiae were obtained using five different condition extraction (GB1: Water; GB1p: Water/proteolysis; GB1s: Water/sonication; GB1sp: Water/sonication/proteolysis; GB2s: NaOH/sonication; GB2sp: NaOH/sonication/proteolysis. The yield (g) increased in the following order GB2sp>GB1sp>GB1p>GB2s>GB1s>GB1. However, the amount of SP extracted increased in different way GB2sp>GB1p>GB1>GB1sp>GB1s>GB2s. Infrared and electrophoresis analysis showed that all conditions extracted the same SP. In addition, monosaccharide composition showed that ultrasound promotes the extraction of other polysaccharides than SP. In the prothrombin time (PT) test, which evaluates the extrinsic coagulation pathway, none of the samples showed anticoagulant activity. While in the activated partial thromboplastin time (aPTT) test, which evaluates the intrinsic coagulation pathway, all samples showed anticoagulant activity, except GB2s. The aPTT activity decreased in the order of GB1sp>GB2sp>GB1p>GB1>GB1s>GB2s. Total capacity antioxidant (TCA) of the SP was also affected by condition extraction, since GB2s and GB1 showed lower activity in comparison to the other conditions. In conclusion, the conditions of SP extraction influence their biological activities and chemical composition. The data showed NaOH/sonication/proteolysis was the best condition to extract anticoagulant and antioxidant SPs from Gracilaria birdiae.

Functional expression of a novel Kunitz type protease inhibitor from the human blood fluke Schistosoma mansoni

BACKGROUND: Schistosomes are able to survive for prolonged periods in the blood system, despite continuous contact with coagulatory factors and mediators of the host immune system. Protease inhibitors likely play a critical role in host immune modulation thereby promoting parasite survival in this extremely hostile environment. Even though Kunitz type serine protease inhibitors have been shown to play important physiological functions in a range of organisms these proteins are less well characterised in parasitic helminths.

METHODS: We have cloned one gene sequence from S. mansoni, Smp_147730 (SmKI-1) which is coded for single domain Kunitz type protease inhibitor, E. coli-expressed and purified. Immunolocalisation and western blotting was carried out using affinity purified polyclonal anti-SmKI-1 murine antibodies to determine SmKI-1 expression in the parasite. Protease inhibitor assays and coagulation assays were performed to evaluate the functional roles of SmKI-1.

RESULTS: SmKI-1 is localised in the tegument of adult worms and the sub-shell region of eggs. Furthermore, this Kunitz protein is secreted into the host in the ES products of the adult worm. Recombinant SmKI-1 inhibited mammalian trypsin, chymotrypsin, neutrophil elastase, FXa and plasma kallikrein with IC50 values of 35 nM, 61 nM, 56 nM, 142 nM and 112 nM, respectively. However, no inhibition was detected for pancreatic elastase or cathepsin G. SmKI-1 (4 μM) delayed blood clot formation, reflected in an approximately three fold increase in activated partial thromboplastin time and prothrombin time.

CONCLUSIONS: We have functionally characterised the first Kunitz type protease inhibitor (SmKI-1) from S. mansoni and show that it has anti-inflammatory and anti-coagulant properties. SmKI-1 is one of a number of putative Kunitz proteins in schistosomes that have presumably evolved as an adaptation to protect these parasites from the defence mechanisms of their mammalian hosts. As such they may represent novel vaccine candidates and/or drug targets for schistosomiasis control.

Homogalactanas sulfatadas da alga Codium isthmocladum com atividade anticoagulante

Since the first description of sulfated polysaccharides from seaweeds, the biological activities of these compounds have been evaluated under different aspects and experimental procedures. Among the broad biological activities presented by seaweed polysaccharides, anticoagulant action appears as a promising function. In this present study we have obtained sulfated polysaccharides from the green seaweed Codium isthmocladium by proteolytic digestion, followed by separation into five fractions (0.3, 0.5, 0.7, 0.9 and 1.2) by sequential acetone precipitation. The chemical analyses have demonstrated that all fractions are composed mainly by sulfated polysaccharides. The anticoagulant activity of these fractions was determined by activated partial thromboplastin time (aPTT) and prothrombin time test (PT) using citrate normal human plasma. None fraction has shown anticoagulant activity by PT test. Furthermore, all of them have shown anticoagulant activity by aPTT test. These results indicated that the molecular targets of these sulfated polysaccharides are mainly in the intrinsic via of the coagulation cascade. Agarose gel electrophoresis in 1,3-diaminopropane acetate buffer, pH 9.0, stained with 0.1% toluidine blue showed the presence of two or three bands in several fractions while the fraction 0.9 showed a single spot. By anion exchange chromatography, the acid polysaccharides from the 0.9 acetone fraction were separated into two new fractions eluted respectively with 2.0 and 3.0 M NaCl. These compounds showed a molecular weight of 6.4 and 7.4 kDa respectively. Chemical analyses and infrared spectroscopy showed that Gal 1 and Gal 2 are sulfated homogalactans and differ one from the other in degree and localization of sulfate groups. aPPT test demonstrated that fractions 2,0 and 3,0M (Gal1 and Gal 2, respectively) have anticoagulant activity. This is the first time that anticoagulant sulfated homogalatans have been isolated from green algae. To prolong the coagulation time to double the baseline value in the aPTT, the required amount of sulfated galactan 1 (6,3mg) was similar to low molecular heparin Clexane®, whereas only 0,7mg of sulfated galactan 2 was needed to obtain the same effect. Sulfated galactan 2 in high doses (250mg) induces platelet aggregation. These results suggest that these galactans from C. isthmocladum have a potential application as an anticoagulant drug

Tenecteplase and alteplase in acute ischaemic stroke thrombolysis: clinical and imaging study

Introduction: Intravenous thrombolysis in acute ischaemic stroke with alteplase improves clinical outcomes, but it has limited efficacy and is associated with increased risk of intracranial haemorrhage. An improved tissue plasminogen activator, tenecteplase, was evidenced to be at least equally effective with lower risk of haemorrhage in acute myocardial infarction thrombolysis. To date, two completed phase II randomised controlled studies comparing tenecteplase and alteplase in acute ischaemic strokes showed variable results. Methods: A literature review of thrombolytic agents used in myocardial infarction and acute ischaemic stroke was performed, followed by a retrospective investigation of the bolus-to- infusion delay of alteplase administration. The main focus of this thesis is the report of our single centre phase II randomised controlled trial that compared tenecteplase (0.25mg/kg, maximum 25mg) and alteplase (0.9mg/kg, maximum 90mg, 10% as the initial bolus, following by one hour infusion with the rest of the dose) in acute ischaemic stroke thrombolysis using advanced imaging as biomarkers. Imaging comprised baseline computed tomography (CT), CT perfusion (CTP) and CT angiography (CTA), and CT+CTA at 24-48 hours. The primary end-point was penumbral salvage (CTP-defined penumbra volume minus follow-up CT infarct volume). A sub-study of coagulation and fibrinolysis analysis of the two agents was performed by comparing a group of coagulation variables measured pre-treatment, 3-12 hours, and 24±3 hours post thrombolysis. An individual patient data (IPD) meta-analysis was carried out using all three completed tenecteplase/alteplase comparison studies in stroke thrombolysis. We compared clinical outcomes including modified Rankin scale at 3 months, early neurological improvement at 24 hours, intracerebral haemorrhage rate and mortality at 3 months between all three tenecteplase doses (0.1mg/kg, 0.25 mg/kg, and 0.4mg/kg) examined and standard alteplase. Imaging outcomes including penumbra salvage, recanalisation rates were also compared using the data from the two studies that had advance imaging carried out. Results: Delay between the initial bolus and the subsequent infusion in administration of alteplase is common. This may reduce the likelihood of achieving a good functional outcome. Among the 104 patients recruited in ATTEST trial, 71 contributed to the imaging primary outcome. No significant differences were observed for penumbral salvage [68 (SD 28) % tenecteplase vs 68 (SD 23) % alteplase], mean difference 1% (95% confidence interval -10%, 12%, p=0·81) or for any secondary end-point. The SICH incidence (1/52, 2% vs 2/51, 4%, by SITS-MOST definition, p=0·55; by ECASS-2 definition, 3/52, 6% tenecteplase vs 4/51, 8% alteplase, p=0.59) did not differed significantly. There was a trend towards lower ICH risk in the tenecteplase group (8/52 tenecteplase, 15% vs 14/51 alteplase, 29%, p=0·091). Compared to baseline, alteplase caused significant hypofibrinogenaemia (p=0.002), prolonged Prothrombin Time (PT) (p=0.011), hypoplasminogenaemia (p=0.001) and lower Factor V (p=0.002) at 3-12 hours after administration with persistent hypofibrinogenaemia at 24h (p=0.011), while only minor hypoplasminogenaemia (P=0.029) was seen in the tenecteplase group. Tenecteplase consumed less plasminogen (p<0.001) and fibrinogen (p=0.002) compared with alteplase. In a pooled analysis, tenecteplase 0.25mg/kg had the greatest odds to achieve early neurological improvement (OR [95%CI] 3.3 [1.5, 7.2], p=0.093), excellent functional outcome (mRS 0-1) at three months (OR [95%CI] 1.9 [0.8, 4.4], p= 0.28), with reduced odds of ICH (OR [95%CI] 0.6 [0.2, 1.8], P=0.43) compared with alteplase. Only 19 patients were treated with tenecteplase 0.4mg/kg, which showed increased odds of SICH compared with alteplase (OR [95% CI] 6.2 [0.7, 56.3]). In the two studies where advanced imaging was performed, the imaging outcomes did not differ in the IPD analysis. Conclusion: Tenecteplase 0.25 mg/kg has the potential to be a better alternative to alteplase. It can be given as a single bolus, does not cause disruption to systemic coagulation, and is possibly safer and more effective in clot lysis. Further phase III study to compare tenecteplase and alteplase in acute ischaemic stroke is warranted.

Aspectos estruturais, farmacológicos e biológicos de fucanas da alga marrom sargassum vulgare

The present study examines the chemical composition and their effects on free radicals, inflammation, angiogenesis, coagulation, VEGF effects and cellular proliferation of a polysaccharides from alga Sargassum vulgare. The sulfated polysaccharide was extracted from brown seaweed by proteolysis with enzymes maxataze. The presence of proteins and sugars were observed in crude polysaccharides. Fractionation of this crude extract was made with growing concentration of acetone (0.3-1.5 v) and produced four groups of polysaccharides. Anionic polysaccharides from brown seaweed Sargassum vulgare, SV1and PSV1 were fractionated (SV1) and purified (PSV1), and displayed with high total sugars and sulfate content and very low level of protein. This fucan SV1 contains low levels of protein and high carbohydrate and sulfate content. This polysaccharides prolonged activated partial thromboplastin time (aPTT) at 50 μg (>240 s). SV1 was found to have no effect on prothrombin time (PT), corresponding to the extrinsic pathway of coagulation. SV1 exhibits high antithrombotic action in vivo, with a concentration ten times higher than heparin. Polysaccharides from S. vulgare promoted direct inhibition enzymatic activity of thrombin and stimulated enzymatic activity of FXa. SV1 showed optimal inhibitory activity of thrombin (50.2±0.28%) at a concentration of 25 μg/mL. Its antioxidant action on scavenging radicals by DPPH was (22%), indicating the polymer has no cytotoxic action (hemolytic) on ABO and Rh blood types in different erythrocyte groups and displays strong anti-inflammatory action on all concentrations tested in the carrageenan-induced paw edema model, demonstrated by reduced edema and cellular infiltration. Angiogenesis is a dynamic process of proliferation and differentiation. It requires endothelial proliferation, migration, and tube formation. In this context, endothelial cells are a preferred target for several studies and therapies. The antiangiogenic efficacy of polysaccharides was examined in vivo in the chick chorioallantoic membrane (CAM) model by using fertilized eggs. Decreases in the density of the capillaries were assessed and scored. The results showed that SV1 and PSV1 have an inhibitory effect on angiogenesis. These results were also confirmed by inhibition tubulogenesis in rabbit aorta endothelial cell (RAEC) in matrigel. These compounds were assessed in Apoptosis assay (Annexin V - FITC / PI) and cell viability by MTT assay of RAEC. These polysaccharides do not affect the viability and do not have apoptotic or necrotic action. RAEC cell when incubated with SV1 and PSV1showed inhibition of VEGF secretion, observed when compounds were incubated at 25, 50 and 100 μg/μL. The VEGF secretion with the RAEC cell line for 24 h, was more effective for PSV1 at 50 μg/μL(71.4%) than SV1 100 μg/μL (75.9%). SV1 and PSV1 had an antiproliferative action (47%) against tumor cell line HeLa. Our results indicate that these sulfated polysaccharides have antiangiogenic and antitumoral actions

Caracterização estrutural e avaliação das atividades farmacológicas da fucana B extraída da alga Dictyota menstrualis

Seaweeds are a major source of biologically active compounds . In the extracellular matrix of these organisms are sulfated polysaccharides that functions as structural components preventing it against dehydration. The fraction 0.9 (FucB) rich in sulfated fucans obtained from brown seaweed Dictyota menstrualis was chemical characterized and evaluated for pharmacological activity by testing anticoagulant activity, stimulatory action on the synthesis of an antithrombotic heparan sulfate, antioxidant activity and its effects in cell proliferation. The main components were FucB carbohydrates (49.80 ± 0.10 %) and sulfate (42.30 ± 0.015 %), with phenolic compounds ( 3.86 ± 0.016 %) and low protein contamination ( 0.58 ± 0.001 % ) . FucB showed polydisperse profile and analysis of signals in the infrared at 1262, 1074 and 930 cm -1 and 840 assigned to S = O bonds sulfate esters , CO bond presence of 3,6- anhydrogalactose , β -D- galactose non- sulfated sulfate and the axial position of fucose C4 , respectively. FucB exhibited moderate anticoagulant activity , the polysaccharides prolonged time (aPTT ) 200 ug ( > 90s ) partial thromboplastin FucB no effect on prothrombin time (PT), which corresponds to the extrinsic pathway of coagulation was observed. This stimulation promoted fraction of about 3.6 times the synthesis of heparan sulfate (HS) by endothelial cells of the rabbit aorta ( RAEC ) in culture compared with cells not treated with FucB . This has also been shown to compete for the binding site with heparin. The rich fraction sulfated fucans exhibited strong antioxidant activity assays on total antioxidant (109.7 and 89.5 % compared with BHT and ascorbic acid standards ) , reducing power ( 71 % compared to ascorbic acid ) and ferric chelation ( 71 , comparing with 5 % ascorbic acid). The fraction of algae showed cytostatic activity on the RAEC cells revealed that the increase of the synthesis of heparan sulfate is not related to proliferation. FucB showed antiproliferative action on cell lines modified as Hela and Hep G2 by MTT assay . These results suggest that FucB Dictyota menstrualis have anticoagulant , antithrombotic , antioxidant potential as well as a possible antitumor action, promoting the stimulation of the synthesis of antithrombotic HS by endothelial cells and is useful in the prevention of thrombosis, also due to its inhibitory action on species reactive oxygen ( ROS ) in some in vitro systems , being involved in promoting a hypercoagulable state

Homogalactanas sulfatadas da alga Codium isthmocladum com atividade anticoagulante

Since the first description of sulfated polysaccharides from seaweeds, the biological activities of these compounds have been evaluated under different aspects and experimental procedures. Among the broad biological activities presented by seaweed polysaccharides, anticoagulant action appears as a promising function. In this present study we have obtained sulfated polysaccharides from the green seaweed Codium isthmocladium by proteolytic digestion, followed by separation into five fractions (0.3, 0.5, 0.7, 0.9 and 1.2) by sequential acetone precipitation. The chemical analyses have demonstrated that all fractions are composed mainly by sulfated polysaccharides. The anticoagulant activity of these fractions was determined by activated partial thromboplastin time (aPTT) and prothrombin time test (PT) using citrate normal human plasma. None fraction has shown anticoagulant activity by PT test. Furthermore, all of them have shown anticoagulant activity by aPTT test. These results indicated that the molecular targets of these sulfated polysaccharides are mainly in the intrinsic via of the coagulation cascade. Agarose gel electrophoresis in 1,3-diaminopropane acetate buffer, pH 9.0, stained with 0.1% toluidine blue showed the presence of two or three bands in several fractions while the fraction 0.9 showed a single spot. By anion exchange chromatography, the acid polysaccharides from the 0.9 acetone fraction were separated into two new fractions eluted respectively with 2.0 and 3.0 M NaCl. These compounds showed a molecular weight of 6.4 and 7.4 kDa respectively. Chemical analyses and infrared spectroscopy showed that Gal 1 and Gal 2 are sulfated homogalactans and differ one from the other in degree and localization of sulfate groups. aPPT test demonstrated that fractions 2,0 and 3,0M (Gal1 and Gal 2, respectively) have anticoagulant activity. This is the first time that anticoagulant sulfated homogalatans have been isolated from green algae. To prolong the coagulation time to double the baseline value in the aPTT, the required amount of sulfated galactan 1 (6,3mg) was similar to low molecular heparin Clexane®, whereas only 0,7mg of sulfated galactan 2 was needed to obtain the same effect. Sulfated galactan 2 in high doses (250mg) induces platelet aggregation. These results suggest that these galactans from C. isthmocladum have a potential application as an anticoagulant drug

Variability in the international normalised ratio (INR) in patients with antiphospholipid syndrome and positive lupus anticoagulant: should the INR targets be higher?

El síndrome antifosfolípido es un desorden autoinmune caracterizado por hipercoagulabilidad que requiere terapia anticoagulante como pilar fundamental, siendo la warfarina el tratamiento de elección en los casos que requieren manejo por largos periodos. Sin embargo, los pacientes con anticoagulante lúpico positivo representan un reto porque tienen mayor riesgo de presentar eventos trombóticos, sumado a que el seguimiento con el International Normalized Ratio (INR) no es confiable, ya que estos anticuerpos generan interferencia con las pruebas de laboratorio basadas en fosfolípidos, como es el caso del tiempo de protrombina (PT) con INR basal prolongado, incluso antes del inicio de la terapia anticoagulante. Por tal razón, se ilustra el caso de una paciente con síndrome antifosfolípido primario y anticoagulante lúpico positivo quien ha presentado múltiples episodios trombóticos, a pesar de recibir terapia anticoagulante. Además se hace una revisión de la literatura disponible y se postulan nuevas metas de INR en estos pacientes diferentes de las que se plantean actualmente.

Reversal of the anticoagulant and anti-hemostatic effect of low molecular weight heparin by direct prothrombin activation

Lopap, found in the bristles of Lonomia obliqua caterpillar, is the first exogenous prothrombin activator that shows serine protease-like activity, independent of prothrombinase components and unique lipocalin reported to interfere with hemostasis mechanisms. To assess the action of an exogenous prothrombin activator reversing the anticoagulant and antihemostatic effect induced by low molecular weight heparin (LMWH), male New Zealand rabbits (N = 20, weighing 3.8-4.0 kg) allocated to 4 groups were anticoagulated with 1800 IU/kg LMWH (iv) over 2 min, followed by iv administration of saline (SG) or recombinant Lopap (rLopap) at 1 µg/kg (LG1) or 10 µg/kg (LG10), 10 min after the injection of LMWH, in a blind manner. Control animals (CG) were treated only with saline. The action of rLopap was assessed in terms of activated partial thromboplastin time (aPTT), prothrombin fragment F1+2, fibrinogen, and ear puncture bleeding time (BT) at 5, 10, 15, 17, 20, 30, 40, 60, and 90 min after initiation of LMWH infusion. LG10 animals showed a decrease of aPTT in more than 50% and BT near to normal baseline. The level of prothrombin fragment F1+2 measured by ELISA had a 6-fold increase with rLopap treatment (10 µg/kg) and was inversely proportional to BT in LMWH-treated animals. Thus, Lopap, obtained in recombinant form using E. coli expression system, was useful in antagonizing the effect of LMWH through direct prothrombin activation, which can be a possible strategy for the reversal of bleeding and anticoagulant events.

Reversal of the anticoagulant and anti-hemostatic effect of low molecular weight heparin by direct prothrombin activation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)