Anticoagulant Effect Of Unfractionated Heparin Research Articles

Thromboelastography versus thromboelastometry for unfractionated heparin monitoring in adult patients on extracorporeal membrane oxygenation.

Background: Monitoring the anticoagulant effect of unfractionated heparin (UFH) in extracorporeal membrane oxygenation (ECMO) patients is complex but critically important to balance the risks of treatment related bleeding and circuit thrombosis. While guidelines recommend using more than one method to monitor UFH activity, the use of thromboelastometry (ROTEM) to monitor UFH in ECMO patients has not been investigated in detail.Methods: This is an observational, single-center retrospective study looking at adult ECMO patients on UFH that had ROTEM and thromboelastography (TEG) tests obtained concurrently. A total of 20 samples were obtained from nine patients during the study period, seven of which were on veno-arterial (VA) ECMO and two of which were on veno-venous (VV) ECMO.Results: Under institutional standard operating practice, when TEG and/or activated partial thromboplastin time (aPTT) were considered therapeutic, intrinsic thromboelastometry clotting time (INTEM CT) was only 1.2 times higher than the normal range. TEG based monitoring compared to aPTT based monitoring tended to result in lower anti-Xa levels and less intensive anticoagulation. For the total cohort, bleeding events, driven by the need for blood transfusions, were more common compared to ischemic events (77% vs 11%; p = 0.02).Conclusion: INTEM CT tended to be less sensitive to lower doses of UFH with a value of 1.2 times higher than the normal range when aPTT and/or TEG were considered therapeutic. Due to the relative insensitivity of ROTEM, our institution decided to continue to use TEG instead of ROTEM. Larger, multicenter trials may be helpful to validate these findings.

Andexanet Alfa Neutralizes the Anticoagulant Effects of Unfractionated Heparin of Bovine, Ovine and Porcine Origin Almost as Protamine Sulfate.

Andexanet alfa (AA) - zhzo, recombinant coagulation factor Xa, is an approved antidote for oral Xa inhibitors (apixaban and rivaroxaban). Unfractionated heparin (UFH) is commonly used for therapeutic, interventional, and surgical indications. Protamine sulfate (PrSO4) is frequently used to neutralize UFH. This study aimed to investigate the comparative neutralization profiles of AA and PrSO4 for heparins of bovine, ovine, and porcine origin. The neutralization effect of PrSO4 at 25 µg/ml and AA at 100 µg/ml was studied on an approximate surgical/interventional concentration of heparin by supplementing whole blood with each of the heparins at 25 µg/ml. For the clotting profile (activated partial thromboplastin time: aPTT), amidolytic (anti-Xa and anti-IIa), and thrombin generation assay each of the heparin were supplemented from -10-0.62 µg/ml. In the whole blood ACT studies, all three heparins produced strong anti-coagulant effects (400-450 seconds) compared to saline (130-150 seconds). Both AA and PrSO4 almost fully neutralized the anti-coagulant effects of heparins (140-160 seconds). Both antidotes completely reversed the anticoagulant effects of all three heparins in the aPTT and thrombin generation assay. However, PrSO4 was more effective in neutralizing the anti-Xa, and anti-IIa effects than AA, which only partially neutralized these effects. Andexanet alfa at 100 µg/ml effectively neutralizes the therapeutic and surgical/interventional concentrations of heparins in in-vitro settings. While differences in the anti-Xa, and anti-IIa effects between heparins were noted, anti-coagulant effect of these agents in the aPTT assay were comparable. A similar neutralization profile was observed in the ACT and thrombin generation assays by both agents.

Anticoagulation Monitoring with Activated Partial ThromboPlastin Time and Anti-Xa Activity in Intensive Care Unit Patients: Interest of Thrombin Generation Assay.

Current guidelines recommend monitoring the anticoagulant effect of unfractionated heparin (UFH) by measuring anti-Xa activity rather than activated partial thromboplastin time (aPTT) in intensive care unit (ICU) patients. The primary objective of this study was to evaluate the correlation of aPTT, anti-Xa activity, and thrombin generation in UFH-treated ICU patients. A prospective observational pilot study was conducted in adult surgical ICU patients treated with UFH. aPTT and anti-Xa activity were monitored daily. The therapeutic target was aPTT between 50 s and 84 s, and/or anti-Xa between 0.3 and 0.7 U/mL. Correlation among aPTT, anti-Xa activity, and thrombin generation was determined by measuring endogenous thrombin potential (ETP), with the inflammatory response evaluated. C-reactive protein (CRP) was used as a marker of inflammatory response. The plasma of 107 samples from 30 ICU patients was analyzed. The correlation between aPTT and anti-Xa activity was 0.66, CI95% [0.54;0.76] (p < 0.0001). Although thrombin generation, aPTT, and anti-Xa were correlated with inflammatory responses, the correlation was higher with thrombin generation and anti-Xa activity compared to aPTT. When aPTT was in a therapeutic range, a low thrombin generation was observed but was 50% inhibited when anti-Xa was in a therapeutic range. Coagulation testing with aPTT, anti-Xa correlated with thrombin generation. A 50% decrease in thrombin generation was observed when anti-Xa was within a therapeutic range. Further work is needed to evaluate coagulation biomarker responses and clinical outcomes in specific ICU populations.

The Development of the Heparin Monitoring System Based on Microfluidics Technology

Heparin monitoring is widely used to measure the anticoagulant effect of unfractionated heparin and adjust the dose to keep within the target treatment range. This technology has applications in many fields and also prospects in the future. Its application has the advantages of rapidity, high throughput and minimum sample consumption. Many point of care devices for heparin monitoring are available. The CoaguChek device only requires a small sample size, which is obtained through a fingerstick. Over the last few years, the point-of-care (POC) testing was used widely for its convenience, efficiency, and faster turnaround times.

The Inhibitory Effect of Protamine on Platelets is Attenuated by Heparin without Inducing Thrombocytopenia in Rodents.

Protamine sulfate (PS) is a polycationic protein drug obtained from the sperm of fish, and is used to reverse the anticoagulant effect of unfractionated heparin (UFH). However, the interactions between PS, UFH, and platelets are still not clear. We measured the platelet numbers and collagen-induced aggregation, P-selectin, platelet factor 4, β-thromboglobulin, prostacyclin metabolite, D-dimers, activated partial thromboplastin time, prothrombin time, anti-factor Xa, fibrinogen, thrombus weight and megakaryocytopoiesis in blood collected from mice and rats in different time points.. All of the groups were treated intravenously with vehicle, UFH, PS, or UFH with PS. We found a short-term antiplatelet activity of PS in mice and rats, and long-term platelet-independent antithrombotic activity in rats with electrically-induced thrombosis. The antiplatelet and antithrombotic potential of PS may contribute to bleeding risk in PS-overdosed patients. The inhibitory effect of PS on the platelets was attenuated by UFH without inducing thrombocytopenia. Treatment with UFH and PS did not affect the formation, number, or activation of platelets, or the thrombosis development in rodents.

A Factor Xa Inhibitor Antidote (andexanet alfa) is Capable of Neutralizing the Anticoagulant Effects of Unfractionated Heparin of Bovine, Ovine and Porcine Origin in a Comparable Manner as Protamine Sulfate

IntroductionAndexanet alfa (zhzo, coagulation factor Xa recombinant) is approved for such factor Xa inhibitors as apixaban and rivaroxaban. Unfractionated heparin (UFH) is commonly used for therapeutic, interventional and surgical indications. Protamine sulfate is commonly used to neutralize UFH. The study investigated the comparative neutralization profiles of andexanet alfa and protamine sulfate for heparins of bovine, ovine or porcine origin.Materials and MethodPowdered form of pharmaceutical grade heparins of bovine (140 U/mg), ovine (200 U/mg) and porcine (190 U/mg) origin were obtained from commercial vendors. Andexanet alfa was obtained from the hospital pharmacy (Skokie Hospital, Skokie, Illinois) and reconstituted at 10 mg/ml according to the manufacturer's instructions. Powdered protamine sulfate (USP grade) was reconstituted at 1.0 mg/ml in saline. To investigate the neutralization profile of these two antidotes for various heparins, each of the bovine, ovine and porcine heparins was supplemented to plasma over a concentration range of 0–10 ug/ml. The effect of protamine sulfate at 10 ug/ml was studied in the activated partial thromboplastin time (aPTT), anti‐Xa and anti‐IIa assays. Similar studies were carried out with andexanet alfa at a concentration of 100 ug/ml. The neutralization effect of protamine sulfate and andexanet alfa was also studied on an approximate surgical/interventional concentration of heparin by supplementing freshly drawn whole blood from healthy individuals (n=5–10) with each of the heparins at a concentration of 25 ug/ml. The neutralization effect of andexanet alfa was studied at 100 ug/ml and protamine sulfate was studied at 25 ug/ml.ResultsBoth andexanet alfa (100 ug/ml) and protamine sulfate (10 ug/ml) completely neutralized the anticoagulant effects of all three heparins in the aPTT assay. However, protamine sulfate was more effective in neutralizing the amidolytic anti‐Xa and anti‐IIa effects in comparison to andexanet alfa which only partially neutralized these effects. In the whole blood clotting ACT studies, all three heparins produced a strong anti‐coagulant effects (400–450 seconds) in comparison to saline (130–150 seconds). Both andexanet alfa (100 ug/ml) and protamine sulfate (25 ug/ml) almost completely neutralized the anti‐coagulant effects of heparins (140–160 seconds).ConclusionThese results clearly suggest that andexanet alfa at 100 ug/ml is effective in neutralizing both the therapeutic and surgical/interventional concentrations of heparins in the in‐vitro settings. While differences in the anti‐Xa and anti‐IIa effects between heparins are noted, the anti‐coagulant effect of these agents in the aPTT assay were comparable and similar neutralization profile was observed in the ACT assays by both agents. The pharmacologic implication of these observations require further investigations.Support or Funding InformationN/AThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Małopłytkowość indukowana protaminą — nowy problem czy inny typ małopłytkowości poheparynowej?

Protamine is widely used to neutralize the anticoagulant effects of unfractionated heparin, particularly after cardiac surgery. Recent reports suggest that some patients undergoing cardiopulmonary bypass exposed to heparin and protamine may develop thrombocytopenia. Multimolecular protamine-heparin complexes lead to immunization and production of immunoglobulin G class antibodies that may activate platelets through FcγIIa receptors. Some patients injected with protamine during cardiopulmonary bypass have increased the risk of early thromboembolic events due to the presence of these antibodies in the blood. In the present review, we will focus on studies investigating the mechanism of platelet activation by anti-protamine-heparin antibodies. We will compare antibodies that are associated with immune heparin-induced thrombocytopenia. In addition, we will describe the clinical consequences of protamine-induced thrombocytopenia, risk factors and general guidelines for management of thromboembolic complications in the post-cardiac surgery patients.

Inadvertent Overdose of Low-Molecular-Weight Heparin in an Elderly Patient with Deep Vein Thrombosis and Acute Kidney Injury.

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Heparin-binding copolymer reverses effects of unfractionated heparin, enoxaparin, and fondaparinux in rats and mice

The parenteral anticoagulants may cause uncontrolled and life-threatening bleeding. Protamine, the only registered heparin antidote, is partially effective against low-molecular weight heparins, completely ineffective against fondaparinux and may cause unacceptable toxicity. Therefore, we aimed to develop a synthetic compound for safe and efficient neutralization of all parenteral anticoagulants. We synthesized pegylated PMAPTAC block copolymers, and then, we selected a lead heparin-binding copolymer (HBC). We assessed the effectiveness of HBC in the model of arterial thrombosis electrically induced in the carotid artery of rats by measuring thrombus weight, bleeding time, activated partial thromboplastin time, activated clotting time, and anti-factor Xa activity. The intravital tissue distribution, the cardiorespiratory, and organ toxicity were monitored. HBC diminished antithrombotic and anticoagulant effects of unfractionated heparin. Moreover, it stopped bleeding and completely reversed the enhancement of clotting times and anti-factor Xa activity caused by enoxaparin or fondaparinux. We observed slight pulmonary congestion and cell infiltration, but the cardiorespiratory parameters remained unchanged. We found a strong signal of fluorescently-labeled HBC in the urine, and a weaker in the liver and in the kidney. No signs of hepatic or nephrotoxicity were observed in the blood biochemistry or histopathologic examination. We developed a copolymer efficiently neutralizing effects of heparins in the living organism, which shows a very promising efficacy/safety profile and may help in the management of uncontrolled bleeding resulting from an anticoagulant injection. HBC could enable the safe replacement of unfractionated heparin with low-molecular weight heparins in patients undergoing cardiac surgery and complex vascular procedures.

Absence of pharmacodynamic interaction between inclacumab and heparin in healthy smokers.

Inclacumab is a novel monoclonal antibody directed against P-selectin in development for the prevention and treatment of atherosclerotic cardiovascular diseases. It is likely to be used concomitantly with heparin in patients undergoing percutaneous coronary intervention. Coadministration of both drugs may potentially increase the bleeding risk associated with heparin. This crossover study evaluated the potential pharmacodynamic interaction between inclacumab and unfractionated heparin in 18 healthy smokers. Owing to the long elimination of inclacumab (half-life of approximately 18 days), a 2-period, one-sequence study design was used. Subjects received an intravenous bolus injection of unfractionated heparin (5000 IU) on days 1 and 8 and an intravenous infusion of inclacumab (20 mg/kg) on day 8. Blood samples were collected on days 1 and 8 for pharmacodynamic effects of unfractionated heparin (anti-FXa and anti-FIIa activities, activated partial thromboplastin time and tissue factor pathway inhibitor) and over 6 months for pharmacokinetics of inclacumab. Sixteen subjects were eligible for pharmacodynamic analysis. Inclacumab had no clinically significant pharmacodynamic interaction with unfractionated heparin. With the exception of the minor but statistically significant increase of the maximum effect [Emax] of anti-FIIa activity, pharmacodynamic parameters (areas under the effect curve [AUElast] and Emax of anti-FXa) were almost similar on days 1 and 8. The 90% confidence intervals of geometric mean ratios of day 8 to day 1 for AUElast and Emax were however all contained within bioequivalence boundaries. The data demonstrate that the anticoagulant effect of unfractionated heparin was not affected by the administration of inclacumab.

Analysis of Anticoagulant Effect of Unfractionated Heparin by Using Thrombin Generation Assay in Vitro

Unfractionated heparin (UFH) is widely used in the treatment and prophylaxis of thrombosis. The anticoa‐ gulant effect of UFH is monitored according to activated partial thromboplastin time (APTT). However, there are reports of cases in which hemorrhagic complications develop despite APTT being maintained within the therapeutic range. This indicates there are problems with the monitoring method using APTT during UFH treat‐ ment. To assess the actual anticoagulant effect in UFH therapy using APTT as a monitoring method, we investi‐ gated changes in APTT and thrombin generation (TG) potential in vitro with the addition of UFH to samples from 10 healthy adults. There were large individual differences in the degree of APTT prolongation with the addition of UFH to samples from healthy adults. Furthermore, individual differences in the degree of TG change exceeded those in APTT in 7 subjects with comparable APTT changes caused by UFH (coefficients of varia‐ tion: 2–5% for APTT, 10–50% for TG potential). Thus, with UFH treatment, there are large individual differen‐ ces in the degree of TG inhibition even when APTT is within the appropriate range for coagulation control. These findings provide invaluable information for resolving the problem of hemorrhagic complications during UFH treatment based on the current APTT method.

2-O, 3-O-Desulfated Heparin (ODSH) Mitigates Chemotherapy-Induced Thrombocytopenia (CIT) by Blocking the Negative Paracrine Effect of Platelet Factor 4 (PF4) On Megakaryopoiesis

Abstract 386Thrombocytopenia is a significant complication of myelosuppressive chemotherapy treatments, which are a mainstay in cancer therapy. We and others have previously shown that PF4 is a negative paracrine affecting megakaryocyte number in culture. In murine studies we showed that platelet PF4 levels are inversely related to steady-state platelet counts and is a major contributor to the duration and severity of CIT. Recently, we have shown that PF4 influences both steady-state platelet count and platelet count recovery in pediatric patients treated for acute lymphoblastic leukemia. Pre-clinical studies in murine models suggest that blocking the effect of PF4 (using polyclonal anti-PF4 antibodies) can mitigate the effect of PF4 on megakaryopoiesis and results in a shortened time to platelet count recovery and higher nadir platelet count. Heparin is known to bind PF4 tightly and can clear PF4 from the vascular endothelium. Pre-clinical studies using heparin to mitigate CIT failed to show an effect, but were limited by dose considerations due to the anti-coagulant effect of unfractionated heparin (UFH) and likely by the inability of this highly negative polysaccharide to reach the intramedullary space in high enough amounts to alter available PF4. ODSH does not exhibit the same effect as unfractionated heparin (UFH) in enhancing antithrombin effects and may allow studies of non-anticoagulant pharmacologic effects of heparin. For example, ODSH retains UFH’s anti-inflammatory effects and its ability to bind tightly to PF4. In preliminary results of a clinical trial using ODSH in conjunction with myelosuppressive chemotherapy in patients with metastatic pancreatic cancer, there was a striking lack of clinically significant CIT as will be reported separately at this meeting. This prompted our evaluation of whether ODSH affects CIT and modulates platelet count recovery and whether this is through its interaction with PF4. We first examined the ability of ODSH to prevent the PF4 effect on megakaryopoiesis in vitro. Using a megakaryocyte colony assay, we show that PF4 treatment of PF4null murine megakaryocytes decreases megakaryocyte colony numbers (54±3% control vs. 23±6% PF4-treated, p<0.001) and treatment with ODSH completely blocks the PF4 effect (55±9% ODSH/PF4-treated, p<0.001 vs. PF4-treated). This suggested that the major mechanism by which ODSH prevents CIT is through inhibition of the PF4 effect. We then examined the effect of ODSH on liquid murine bone marrow culture and showed that ODSH treatment (50 μg/mL) was able to improve cell counts in the presence of added recombinant human PF4 (50 μg/mL) (9.2±3.3 × 104 cells/mL in PF4-treated cells vs. 19.3±4.2 × 104 cells/mL in ODSH+PF4-treated cells, p<0.01). Finally, we examined the in vivo effect of ODSH on CIT in a murine model of chemotherapy in transgenic mice that overexpress human (h) PF4. In these hPF4 mice, endogenous PF4 levels significantly affect enhances the degree and duration of 5FU-induced CIT. We previously reported that treatment of animals with anti-hPF4 antibodies was able to completely abolish the PF4 effect. Using the same model, injecting 180 mg/kg 5-fluourouracil (5-FU) intraperitoneally on day 0, we examined the effect on platelet counts of treatment with 2 clinically relevant doses of ODSH (25 mg/kg/dose) given subcutaneously 30 minutes and 24 hours after injection of 5-FU. hPF4 mice treated with 5-FU and ODSH had a higher platelet count nadir (70±14% versus vs. 44±1% of baseline). The nadir platelet count in the ODSH-treated mice was similar to that in mPF4null mice (57±20% of baseline). In addition, animals treated with ODSH recovered approximately 2 days earlier. In summary, ODSH mitigates CIT resulting in decreased severity and duration of thrombocytopenia. These studies suggest that this effect is mediated in large part by PF4 as in vitro experiments show that ODSH completely blocks the effect of PF4 on megakaryopoiesis. The in vivo studies support that sufficient ODSH reaches the marrow to block intramedullary-released PF4 and prevents its inhibition of megakaryopoiesis. This drug is already in clinical trials in humans and may be the first clinically relevant inhibitor of CIT. Further studies will examine its effect in other thrombocytopenic settings. Disclosures:Marcus:Paringenix: Employment, Equity Ownership.

Can Heparin-Based Therapy in Children Be Monitored on Samples Drawn Through Ports? An Analysis From Clinical Trials Data,

Abstract 3365 Background:Based on preliminary data, children clear heparins more rapidly than adults, and thus most pediatric hematologists monitor heparin therapy. Many pediatric deep vein thrombosis patients have indwelling central venous lines (CVLs) due to co-morbid conditions and patients and clinicians desire to use these CVLs for blood sampling. Standard protocols are used to flush and clear CVLs prior to sample collection, but validated approaches to exclude contamination of heparin in dwell-volume (i.e., hep-lock) from systemic anticoagulant have not been established. Aims: This study was conducted to determine the accuracy of heparin monitoring in samples collected from CVLs. Methods: Anticoagulant effects of unfractionated heparin (UH) and dalteparin low molecular weight heparin (D) were determined by assay of anti-IIa activity, anti-Xa activity and the ratio of the two using chromogenic assays (Biophen). Normal pooled plasma was spiked with 2 U/mL of either drug and dilutions were made to 1.5, 1, 0.75, 0.5, 0.25 and 0.1 U/mL in normal pooled plasma. The assays and ratios were applied to plasma samples obtained by standard protocol from CVLs or peripheral venipuncture in two separate clinical trials. Results: Intra-assay CV for anti-IIa was 14.2 and 13.3% for UH and D, respectively; intra-assay CV for anti-Xa was 8.3 and 2.8% for UH and D, respectively. The ratio of anti-IIa to anti-Xa activity for UH was > 0.75 at all concentrations, while the ratio of anti-IIa to anti-Xa for D was < 0.5 when anti-Xa was within the therapeutic range of 0.5–1.0 U/mL, but overlapped with UH at concentrations ≥ 1.5 U/mL and ≤ 0.25 U/mL. In trial 1, DAVINCI, 4/26 (15.4% of) samples drawn from CVLs with therapeutic anti-Xa activity had ratios > 0.5 suggestive of UH contamination, while 0/8 peripherally drawn samples had ratios in that range. In trial 2, 5/26 (19.2% of) patient samples drawn from CVLs had ratios > 0.5. Conclusions: An anti-IIa/anti-Xa ratio < 0.5 is specific for uncontaminated D effect when anti-Xa is within the therapeutic range. However, without measuring ratios 15–20% of CVL samples used to monitor D will overestimate therapeutic response. Therefore, we favor peripherally drawn samples in order to reliably monitor anticoagulation in children. Disclosures:Manco-Johnson:Easai: Research Funding. Goldenberg:Easai: Research Funding.

Monitoring heparin anticoagulation in the acute phase response

The anticoagulant effect of unfractionated heparin (UFH) is monitored using the activated partial thromboplastin time (APTT). An APTT of 1.5-2.5 times the control is usually taken as the therapeutic range and assumed to reflect an anti-activated factor X (anti-Xa) level of 0.35-0.7 u/ml. However, in some cases, despite administration of sufficient heparin to achieve a therapeutic anti-Xa assay level, the APTT remains sub-therapeutic. This 'apparent heparin resistance' is commonly due to high levels of factor VIII (FVIII). In these situations, the anti-Xa is usually preferred for monitoring in order to avoid, what might be, dangerously high levels of heparin. We hypothesized that at high FVIII levels, the heparin resistance encountered may be genuine rather than apparent and that higher doses of heparin may indeed be needed for an equivalent anticoagulant effect. The relationship between heparin level, APTT and anticoagulant effect at different FVIII concentrations was determined using thrombelastography and the thrombin generation assay. Thromboelastographic and thrombin generation parameters concurred with APTT, demonstrating a genuine heparin resistance in the presence of high FVIII levels. This suggests that APTT may be a more accurate measure of anticoagulant effect in vivo than anti-Xa.

Design and rationale of the Evaluation of M118 IN pErcutaNeous Coronary intErvention (EMINENCE) trial

Currently recommended anticoagulant agents used in the setting of percutaneous coronary intervention (PCI) inhibit, with varying degrees of intensity, 2 critical targets (factor Xa and/or IIa) of the coagulation cascade, yet they carry significant limitations. M118-a novel, rationally engineered heparin-provides consistent anti-Xa and anti-IIa activity with a constant anti-Xa:anti-IIa ratio over time. M118 also combines the desired anticoagulant effects of unfractionated heparin with the beneficial attributes of low-molecular-weight heparin, and may represent the next generation of heparin therapy in patients diagnosed with acute coronary syndrome. The EMINENCE trial is a prospective, randomized, open-label, multicenter phase 2 study that will evaluate the safety and feasibility of M118 as an anticoagulant versus unfractionated heparin in subjects with stable coronary artery disease undergoing PCI. The primary end point of the study will be the combined incidence of clinical events defined as the composite of 30-day death, myocardial infarction, repeat revascularization, catheter thrombus, stroke, thrombocytopenia, bailout use of glycoprotein IIb/IIIa inhibitors, and major or minor bleeding. The EMINENCE trial will assess the safety and feasibility of M118 as an anticoagulant in the setting of PCI and will provide important information to determine the appropriate therapeutic range of activated clotting time for M118 and the appropriate dose or doses to be explored in a phase 3 clinical trial.

Is the inhibition of both clot-associated thrombin and factor Xa more clinically relevant than either one alone?

The procoagulant activity of a thrombus is essentially due to clot-associated factor IIa and factor Xa activities.The aim of this review is to underline that specific antithrombin and anti-Xa drugs, such as r-hirudin and DX 9065a, respectively, are complementary, and could be used in combination in clinical trials in patients with acute arterial thrombosis such as coronary syndromes. After standardization of the in-vitro techniques for clot-bound thrombin and clot-associated factor Xa, we have studied the anticoagulant effect of unfractionated heparin and a low-molecular heparin in an in-vitro model. We have confirmed the inability of heparins to inhibit clot-bound thrombin and clot-associated factor Xa. We have compared r-hirudin, a direct thrombin inhibitor, with DX9065a, a direct factor Xa inhibitor. We have observed that r-hirudin inhibited clot-bound thrombin but not clot-bound factor Xa. After r-hirudin treatment interruption, a hypercoagulation rebound has been reported and it could be in relation with the persistence of factor Xa activity in the clot. We have demonstrated that DX9065a inhibits clot-bound factor Xa but does not inhibit clot-bound factor IIa. The complementary effect of DX9065a and r-hirudin is demonstrated in this experimental model. It is likely that several other combinations of drugs may also exhibit an increase of the antithrombotic activity which could be of interest in clinical implication for the treatment of several groups of patients at high risk of arterial thrombosis.

Do we need thrombin generation assays for monitoring anticoagulation?

Thromb Haemost 2008; 100: 179–180 Protamine sulphate neutralizes the anticoagulant effect of unfractionated heparin (UFH), and this effect can easily be demonstrated by a variety of assay systems, including the traditional activated partial thromboplastin time (aPTT), as well as anti-factor Xa assays, or thrombin generation tests such as the Calibrated Automated Thrombogram (CAT) (1). Protamine also neutralizes the anticoagulant effect of low-molecular-weight heparins (LMWH), but to a lesser and variable extent (2). In this issue of Thrombosis and Haemostasis Gatt et al. (3) compared the effect of protamin sulphate and other drugs on the effect of UFH, LMWH, danaparoid and fondaparinux on aPTT, anti-factor Xa-level, and a thrombin generation test. Reversal of anticoagulation is an interesting model for testing the performance of monitoring assays for anticoagulant drugs. One important finding of the present paper by Gatt et al. is that protamine has little effect on apparent anti-factor Xa-activity levels of LMWH, but a considerable effect on the CAT results. This suggests that the success of protamine administration cannot be monitored by measuring anti-factor Xa activity.This is in some contrast to other investigations showing that anti-factor Xa activity of LMWH is affected by protamine (4, 5). Finally, protamine has no effect on the anticoagulant effect of danaparoid and fondaparinux, and this was evident in both anti-factor Xa-activity and CAT assays. Reversal of danaparoid and fondaparinux, and the conventional LMWH is seldom necessary, but these drugs display a considerably prolonged half-life in patients with impaired renal function and severe bleeding may occur if the dose is not adjusted appropriately (6). Monitoring of the anticoagulant effect in patients with impaired renal function and in critically ill patients in general is essential for prevention of bleeding complications, and an assay system that gives a more global image of haemostatic function than an anti-factor Xa assay might be advantageous (7). Recombinant factor VIIa (rFVIIa, NovoSeven®) has been suggested for treatment of patients with severe anticoagulant-induced bleeding (8), and several case reports have been published on the use of rFVIIa in bleeding fondaparinux-treated patients (9, 10). As shown by Gatt et al. (3), the anti-factor Xa levels are not influenced by rFVIIa, whereas the CAT results reflect the procoagulant effect of rFVIIa. FEIBA® is an alternative to rFVIIa in the treatment of haemophilia patients with inhibitors, and may also be helpful in anticoagulant-induced bleeding. In the present experiments of Gatt et al., FEIBA acted quite similar to rFVIIa, although rFVIIa seemed to be more efficient. Another interesting result is that fresh frozen plasma reduces the anti-factor Xa-activity of UFH, LMWH, danaparoid, and fondaparinux, but has little effect on the anticoagulant effect measured by CAT. The main effect of fresh frozen plasma is dilution, and it might be that the threshold concentration of the anticoagulants for an effect on the assay systems is different. Additional experiments might be needed to determine the threshold concentration of LMWH, danaparoid and fondaparinux for the CAT. From the present results, and experiments by other investigators it seems that thrombin generation tests more reliably reflect the anticoagulant effect of these drugs (11). Anticoagulant drugs delay clot formation and traditional methods for the monitoring of anticoagulation are based precisely on this effect.After addition of an activator, clot formation occurs when only about 5% of the prothrombin has been activated to form thrombin. There are many theories why additional thrombin is formed after clot formation.The additional thrombin may not only improve clot stability by activating factor XIII and TAFI, induce cellular invasion or activation of endothelial cells adjacent to the clot in vivo, but also serves to induce the anticoagulant protein C-dependent feedback control of thrombin generation. Thrombin generation assays not only detect delayed thrombin generation (leading to a prolonged clotting time), but also reduced thrombin activity (leading to changes in structural and functional properties of the clot). Both may be relevant for the therapeutic effect of anticoagulant drugs. The results of Gatt et al. (3) indicate that the CAT may be a prototype for global coagulation assays used for monitoring of anticoagulant therapy. New anticoagulants are entering the scene and one of the major advantages of these drugs is the reliable anticoagulant effect with little intraand inter-individual varia-

Heparanase, heparin and the coagulation system in cancer progression

Heparanase is an endoglycosidase which cleaves heparan sulfate (HS) and hence participates in degradation and remodeling of the extracellular matrix (ECM). The enzyme also releases angiogenic factors from the ECM and thereby induces an angiogenic response in vivo. Heparanase is preferentially expressed in human tumors and its over-expression in tumor cells confers an accelerated growth and invasive phenotype in experimental animals. In contrast, heparanase gene silencing is associated with a marked inhibition of tumor progression. Heparanase upregulation correlates with increased tumor vascularity and poor postoperative survival of cancer patients. Studies on relationships between structure and the heparanase-inhibiting activity of nonanticogulant heparins systematically differing in their O-sulfation patterns, degrees of N-acetylation, and glycol-splitting of nonsulfated uronic acid residues, have permitted to select effective inhibitors of the enzymatic activity of heparanase. N-acetylated, glycol-split heparins emerged as highly effective and specific inhibitors of heparanase and tumor growth and metastasis. Several observations support the involvement of heparanase in haemostasis. A marked induction of tissue factor (TF) was noted in response to heparanase over-expression in tumor-derived cell lines and heparanase over-expressing transgenic mice. A direct correlation was also found between heparanase and TF expression levels in leukemia patients. TF induction was even more pronounced upon exogenous addition of heparanase to primary endothelial cells that do not normally express TF, and this induction was associated with enhanced coagulation. These and other results indicate that pro-heparanase is rapidly tethered on cell surfaces, partially depending on cell surface heparan sulfate, generating a local procoagulant effect. In addition, pro-heparanase can reverse the anti-coagulant effect of unfractionated heparin and the Factor Xa inhibitory activity of low molecular weight heparin (LMWH). These effects were also demonstrated in plasma derived from patients treated with LMWH. The pro-coagulant effects of pro-heparanase were also exerted by a peptide corresponding to its major functional heparin-binding domain. Heparanase pro-coagulant activities suggest its possible role as a natural regulator of heparinoid anti-coagulant activities, and point to a possible use of this molecule or its heparin binding domain as antidote for heparinoid therapies.

Monitoring unfractionated heparin with the aPTT: Time for a fresh look

Laboratory monitoring is widely recommended to measure the anticoagulant effect of unfractionated heparin and to adjust the dose to maintain levels in the target therapeutic range. The most widely used laboratory assay for monitoring unfractionated heparin therapy is the activated partial thromboplastin time (aPTT). A fixed therapeutic range for the aPTT of 1.5 to 2.5 times the control value has become widely accepted, but the evidence supporting this range is weak and the clinical validity of using the aPTT for predicting thrombotic or bleeding events is questionable. The aPTT test is also affected by numerous preanalytic and analytic variables that are unrelated to the anticoagulant effect of unfractionated heparin, further eroding its potential value for monitoring unfractionated heparin treatment. Unfractionated heparin dose appears to be more important than the aPTT in predicting clinical efficacy. Despite serious limitations, the reliance on the aPTT is likely to continue because of its ready availability and familiarity of clinicians with the test. The focus of clinicians who manage unfractionated heparin therapy should be to ensure that an adequate starting dose of unfractionated heparin is used and that the aPTT method is standardized. Future research efforts should be directed towards developing methods to improve standardization of the aPTT assay for monitoring unfractionated heparin. Direct measures of the concentration of unfractionated heparin in the blood are attractive because these assays are not affected by many of the biologic variables that interfere with the aPTT and may be suitable for automation. However, currently available unfractionated heparin assays are much more expensive than the aPTT, are not widely available, and their validity has not been adequately assessed in clinical outcome studies.

New Antithrombotic Drugs

For many decades, heparin and oral anticoagulants have been the antithrombotic drugs of choice in the prevention and treatment of both venous and arterial thromboembolism. The efficacy and the relative safety of these compounds have been consistently proven by a large number of clinical studies that have also progressively contributed to improve their management. However, these drugs have limitations. Unfractionated heparin (UFH) acts by catalyzing the effect of antithrombin III, a natural anticoagulant able to inactivate factors IIa, IXa, and Xa, and also by inhibiting the activation of factors V and VIII by thrombin. The anticoagulant effect of UFH is unpredictable and varies considerably among patients, depending upon age, gender, body weight, smoking status, and renal function. This wide variability is caused by heparin binding to phase reactant proteins; the level of which varies among normal individuals and among disease states. Before therapeutic plasma levels are achieved, the binding to plasma proteins as well as to receptor sites on endothelium must be saturated. Unfractionated heparin must be given parenterally and it requires careful laboratory monitoring to ensure the anticoagulant response is therapeutic.1 Low molecular weight heparin (LMWH) also acts by catalyzing antithrombin III, but because of the reduced number of saccharide units, its antithrombotic activity is more selective and it is mainly based on inactivation of factor Xa because of a reduced ability to inactivate factor IIa. Low molecular weight heparin does not bind to the endothelium and have a lower affinity for plasma proteins. This results in a more predictable bioavailability, in a substantially longer half-life, and in a stable dose response when injected subcutaneously. Therefore, LMWH has a more predictable anticoagulant response that has overcome the need for laboratory monitoring. However, it also needs to be administered parenterally and thus has limited indications for long-term use.2 Characteristics and pharmacokinetics of UFH and LMWH are summarized in T1 and T2. In the last years, new antithrombotic agents have been developed to overcome some of heparin and warfarin limitations. One of the main goals was the search for a greater selectivity in the pharmacologic action of the new compounds. Direct factor Xa inhibitors (DTI) and direct thrombin inhibitors recently underwent extensive evaluation in the prevention and treatment of both venous and arterial thromboembolism. science [coagulation and hematology] New Antithrombotic Drugs Walter Ageno,1 Alexander G.G. Turpie2 1Department of Internal Medicine, University of Insubria, Varese, Italy, 2Department of Internal Medicine, McMaster University, Hamilton, Canada