Endogenous heparinoids detected by anti-Xa activity are present in blood during acute variceal bleeding in cirrhosis. A prospective study.

BACKGROUND & AIMS: Endogenous heparinoids have been detected by thromboelastography and quantified by clotting based anti-Xa activity assays in patients with cirrhosis, but their presence in variceal bleeding has not been established yet. METHODS: Clotting based anti-Xa activity was measured in A) 30 cirrhotics with variceal bleeding, B) 15 non-cirrhotics with peptic ulcer bleeding, C) 10 cirrhotics without infection or bleeding, and D) 10 cirrhotics with hepatocellular carcinoma (HCC). RESULTS: Anti-Xa activity was not detected in ulcer bleeders or in cirrhotics without infection or bleeding but was present in seven (23%) variceal bleeders (median levels: 0.03 u/mL (0.01-0.07)) and was quantifiable for 3 days in six of seven patients. Four of seven variceal bleeders with anti-Xa activity present had HCC (p=0.023). Age, creatinine, platelet count and total infections the second day from admission were significantly correlated with the presence of measureable anti-Xa levels (p=0.014, 0.032, 0.004 and 0.019, respectively). In the HCC group, anti-Xa activity was present in three patients (30%) [median levels: 0.05 u/mL (0.01-0.06)]. CONCLUSIONS: In this study, variceal bleeders and 30% of the patients with HCC had endogenous heparinoids that were detected by a clotting based anti-Xa activity assay, whereas there was no anti Xa activity present in patients with cirrhosis without infection, or bleeding or HCC, nor in those with ulcer bleeding. Thus, the anti Xa activity is likely to be a response to bacterial infection and/or presence of HCC in cirrhosis.

Evaluation of coagulation abnormalities in acute liver failure.

BACKGROUND & AIMS: In acute liver failure (ALF), prothrombin time (PT) and its derivative prothrombin time ratio (PTR) are elevated, and are considered predictors of increased bleeding risk. We aimed at determining whether increased PT/PTR reflects the haemostatic potential and bleeding risk in ALF patients. METHODS: Twenty consecutive ALF patients were recruited. Samples were analysed on admission for standard laboratory clotting tests (e.g. PT), thromboelastography (TEG), individual pro and anticoagulant factors and thrombin generation (TG) kinetics with and without Protac, a snake venom protein C activator, and microparticle assay. TG was also measured in 20 age and sex matched healthy volunteers. RESULTS: PT was significantly raised (50.7s ± 7.2, p=0.0001) but did not correlate with TEG parameters. TEG tracings were consistent with a hypocoagulable state in 20%, normal in 45%, and hypercoagulable in 35% of the patients. There was a concomitant and proportional reduction in plasma levels of both procoagulants and natural anticoagulant proteins, in conjunction with a significant elevation in plasma levels of factors-VIII (FVIII) and Von Willebrand factor, and microparticles, culminating in an overall efficient, albeit reduced, thrombin generation capacity in comparison with healthy individuals. A heparin-like effect (HLE) was also noted in most patients. No significant clinical bleeding complications occurred and no blood transfusions were required. CONCLUSIONS: In ALF, despite grossly deranged PT in all patients, estimation of bleeding risk suggests that the coagulation disturbance in ALF patients is complex and heterogeneous for which an individualised approach is required.

Endogenous heparinoids induced massive hemorrhage postoperatively following THA.

A 64-year-old woman presented with right hip pain of 6 years' duration, accompanied by limping of 1 years' duration. Preoperative pelvis radiograph showed narrowing of joint space, osteophyte formation, and cystis degeneration of the femoral head surrounded by bony sclerosis, without obvious collapse of the femoral head; the diagnosis was osteoarthrosis. The patient's blood pressure on admission was 128/76 mm Hg. The patient had no special previous history. Preoperative blood and coagulation tests were normal. Total hip arthroplasty was performed via a posterolateral approach, and intraoperative bleeding was 400 mL. In the first 10 hours postoperatively, the drainage volume decreased slowly, the initial dose of 0.3 mL Fraxiparine was administered, and the blood pressure was 84∼92/45∼56 mm Hg. The drainage volume increased gradually, and 10 hours after administration of Fraxiparine, the drainage volume increased sharply and maintained at a high level. Protamine was applied and the drainage volume decreased sharply, furthermore, a high concentration of endogenous heparinoids were detected in the blood. Although no direct evidence were detected, it was clear that the massive hemorrhage was associated with administration of low-molecular-weight heparin (LMWH). The administration of LMWH combined with continuous low blood pressure caused by surgery and massive bleeding that resulted in low perfusion and inflammation in microcirculation, induced endogenous heparinoid synthesis in endothelial cells, and elevated concentration of endogenous heparinoids in blood led to more severe bleeding. Therefore, caution should be taken with administration of anticoagulant therapy in patients with massive hemorrhage, especially in patients with continuous low blood pressure.

An uncommon cause of postoperative bleeding.

The postoperative phase can be associated with an increased risk of thrombosis and abnormal bleeding. We report the case of a patient admitted to our intensive care unit for postoperative monitoring who developed two episodes of hemorrhagic shock. A thromboelastogram performed with heparinase I digestion demonstrated the action of endogenous heparin-like substances whose presence was subsequently confirmed at the histological examination of the tumor. Heparinase-modified thromboelastography has proven to be an effective and rapid point-of-care coagulation test and in our case allowed early detection of circulating heparinoids.

Comparative study of Factor Xa fluorogenic substrates and their influence on the quantification of LMWHs.

Low molecular weight heparins (LMWHs) are recognised as the preferred anticoagulants in the prevention and treatment of venous thromboembolism. Anti-Factor Xa (anti-FXa) levels are used to monitor the anticoagulant effect of LMWHs and such assays are routinely employed in hospital diagnostic laboratories. In this study, a fluorogenic anti-FXa assay was developed using a commercially available fluorogenic substrate with an attached 6-amino-1-naphthalene-sulfonamide (ANSN) fluorophore and was used for the determination of two LMWHs, enoxaparin and tinzaparin and the heparinoid, danaparoid. The assay was based on the complexation of heparinised plasma with 100 nM exogenous FXa and 25 µM of the fluorogenic substrate Mes-D-LGR-ANSN (C(2)H(5))(2) (SN-7). The assay was tested with pooled plasma samples spiked with anticoagulant concentrations in the range 0-1.6 U mL(-1). The statistically sensitive assay range was 0-0.4 U mL(-1) for enoxaparin and tinzaparin and 0-0.2 U mL(-1) for danaparoid, with assay variation typically below 10.5%. This assay was then compared with a previously published fluorogenic anti-FXa assay developed with the peptide substrate, methylsulfonyl-D: -cyclohexylalanyl-glycyl-arginine-7-amino-4-methylcoumarin acetate (Pefafluor FXa). Both assays were compared in terms of fluorescence intensity, lag times and sensitivity to anticoagulants.

Hepatoenteric ischemia-reperfusion increases circulating heparinoid activity in rabbits.

PURPOSE: The purpose of this study was to determine if an increase in circulating heparinoid activity contributes to the hemostatic abnormalities associated with hepatoenteric ischemia-reperfusion. MATERIALS AND METHODS: Anesthetized rabbit (n = 18) underwent thoracic aorta occlusion for 30 minutes with a balloon catheter, followed by 30 minutes of reperfusion. Blood samples were obtained after 30 minutes of equilibration and 30 minutes of reperfusion. Hemostatic function was assessed by changes in the thrombelastographic variables R (reaction time), alpha (a measure of the speed of clot formation), and G (a measure of clot strength). Thrombelastography was performed on blood without platelet inhibition in the presence or absence of heparinase (n = 9 rabbits). Additional samples (n = 9) were exposed to cytochalasin D (platelet inhibitor) with or without heparinase. RESULTS: Compared with preischemic values, blood samples with intact platelet function obtained during reperfusion demonstrated a decrease in hemostatic function evidenced by a significant (P<.05) increase in R, decrease in alpha, and decrease in G. R, alpha, and G values of samples without platelet inhibition exposed to heparinase did not significantly change after ischemia. Blood samples exposed to cytochalasin D displayed a similar pattern. CONCLUSION: An increase in circulating heparinoid activity significantly contributes to the hemostatic disorder associated with hepatoenteric ischemia-reperfusion in rabbits.

Intraoperative monitoring of danaparoid sodium anticoagulation during cardiovascular operations.

PURPOSE: Patients with cardiovascular disorders frequently need anticoagulation for diagnostic studies, surgical procedures, and therapy. Heparin-induced thrombocytopenia is a relatively common complication of heparin therapy that can result in thrombosis and subsequent limb loss or death, necessitating use of alternative anticoagulants. METHODS: Two patients who needed cardiac surgery had thrombocytopenia induced by exposure to heparin and heparin-coated tubing. Several assays were examined for their ability to monitor intraoperative anticoagulation of a factor Xa inhibitor, danaparoid sodium. RESULTS: In vitro, celite and kaolin activated dotting times and activated partial thromboplastin time were prolonged linearly in the presence of increasing concentrations of danaparoid sodium. Aprotinin did not alter the linearity of the response but did alter its slope. In vivo, activated clotting times and activated partial thromboplastin time were insensitive to clinically significant changes in danaparoid sodium plasma levels during cardiopulmonary bypass. Correction in activated partial thromboplastin time lagged 2 hours behind clinically important changes in anti-factor Xa levels. Only anti-factor Xa levels were adequate to monitor intraoperative danaparoid sodium levels. CONCLUSION: Anticoagulation for cardiopulmonary bypass can be successfully performed with danaparoid sodium and intraoperative anti-factor Xa monitoring.

Quantitative determination of heparinoid mimetics in human and rat plasma by micellar electrokinetic chromatography.

The investigated new class of heparinoid mimetics are spaced persulfated carbohydrates designed to increase the success rate of angioplasty and bypass surgery by preventing restenosis without increasing the risk of bleeding. Due to the presence of sulfate groups, these compounds are highly charged and, for preclinical kinetic investigations only small sample volumes of rat plasma were available. Therefore, capillary electrophoresis (CE) was applied. A bioanalytical method based on micellar elektrokinetic chromatography (MEKC) with UV-detection was developed for the selective quantitation of heparinoid mimetics (e.g., Ro 48-0843, Ro 48-3151, Ro 47-6199 and Ro 48-8722) in plasma. Using this method, only small volumes of plasma were required, which could be introduced directly into the separation capillary after 1:1 dilution with 100 mM aqueous sodium dodecyl sulfate (SDS). For increased sample throughput, an additional ultrafiltration step was performed after SDS-dilution of the plasma sample, improving both reproducibility and robustness of the method considerably. The sensitivity of the new method (3 micrograms/ml) was sufficient to follow plasma levels in pharmacokinetic studies. The practicability of this easy and rapid assay was demonstrated by the analysis of more than 350 plasma samples from pharmacokinetic studies performed in rats.

[A clinical constelation in disuse: vitiligo, mastocytosis of the bone marrow, circulating heparinoid, chronic leg ulcer, hypoalbuminemia, ulcerative colitis and hepatic fibrosis]. / Una constelación clínica desusada: vitíligo, mastocitosis de la médula ósea, heparinoide circulante, úlcera crónica de la pierna, hipoalbuminemia, colitis ulcerosa y fibrosis hepática.

We report an unusual case. Our patient had vitiligo, ulcerative colitis, hepatic fibrosis, hypoalbuminemia, bone marrow mastocytosis, a circulating heparinoid and chronic leg ulcers. The relationship between some of the components of this constellation seems logical. This is the case with bone marrow mastocytosis and circulating heparinoid. Also, between hypoalbuminemia and colitis and liver disfunction. On the other hand, the relationship between other features does not seem clear in the light of current knowledge.