Severity of asphyxia is a covariate of phenobarbital clearance in newborns undergoing hypothermia.

AIM: Phenobarbital (PB) pharmacokinetics (PK) in asphyxiated newborns show large variability, not only explained by hypothermia (HT). We evaluated potential relevant covariates of PK of PB in newborns treated with or without HT for hypoxic-ischemic encephalopathy (HIE). METHODS: Clearance (CL), distribution volume (Vd) and elimination half-life (t1/2) were calculated using one-compartment analysis. Covariates were clinical characteristics (weight, gestational age, hepatic, renal, and circulatory status), comedication and HIE severity [time to reach normal aEEG pattern (TnormaEEG), dichotomous, within 24 h] and asphyxia severity [severe aspyhxia = pH ≤7.1 + Apgar score ≤5 (5 min), dichotomous]. Student's t-test, two-way ANOVA, correlation and Pearson's chi-square test were used. RESULTS: Forty newborns were included [14 non-HT; 26 HT with TnormaEEG <24 h in 14/26 (group1-HT) and TnormaEEG ≥24 h in 12/26 (group2-HT)]. Severe asphyxia was present in 26/40 [5/14 non-HT, 11/14 and 10/12 in both HT groups]. PB-CL, Vd and t1/2 were similar between the non-HT and HT group. However, within the HT group, PB-CL was significantly different between group1-HT and group2-HT (p = .043). ANOVA showed that HT (p = .034) and severity of asphyxia (p = .038) reduced PB-CL (-50%). CONCLUSION: The interaction of severity of asphyxia and HT is associated with a clinical relevant reduced PB-CL, suggesting the potential relevance of disease characteristics beyond HT itself.

Diagnostics and treatment of children with acute seizures on paediatric intensive care unit.

218 children were admitted to paediatric clinic for acute seizures within last 5 years. Out of them, 14 children (7%) were admitted to paediatric intensive care unit (PICU) with repeated or prolonged seizures. Two children were hospitalized twice. The average age of children in time of admission was 12 months, most of the children were healthy before admission, had normal psychomotor development. The most frequent seizure manifestation was asymmetric clonic seizure. Hypoventilation and/or unconsciousness was the most frequent indication for PICU admission in addition to seizures. Febrile seizure was the most frequent diagnosis in children with shorter or less frequent episodes of seizure. Children with severe course and pharmacoresistant seizure were likely to have genetic diagnosis. The MRI scan was normal in most of the children, interictal EEG was mostly without specific finding. Midazolam was the first lime anticonvulsive medication used in most of the children, while phenobarbital, levetiracetam or phenytoin were the preferred second line drugs. Keywords: acute seizures, asymmetric clonic seizure, febrile seizure, children, paediatric intensive care unit.

Neonatal Onset of Epilepsy of Infancy with Migrating Focal Seizures Associated with a Novel GABRB3 Variant in Monozygotic Twins.

BACKGROUND: Recently, a study providing insight into GABRB3 mutational spectrum was published (Møller et al 2017). The authors report considerable pleiotropy even for single mutations and were not able to identify any genotype-phenotype correlations. METHODS: The proband (twin B) was referred for massively parallel sequencing of epilepsy-related gene panel because of hypotonia and neonatal seizures. The revealed variant was confirmed with Sanger sequencing in the proband and the twin A, and both parents were tested for the presence of the variant. RESULTS: We report a case of epilepsy of infancy with migrating focal seizures (EIMFS) of neonatal onset in monozygotic twins with a de novo novel GABRB3 variant p.Thr281Ala. The variant has a uniform presentation on an identical genomic background. In addition, early seizure-onset epilepsy associated with GABRB3 mutation has been until now described only for the p.Leu256Gln variant in the GABRB3 (Møller et al 2017, Myers et al 2016) located in the transmembrane domain just as the p.Thr281Ala variant described here. CONCLUSION: De novo GABRB3 mutations may cause neonatal-onset EIMFS with early-onset hypotonia, respiratory distress, and severe developmental delay.

Addition of platelet concentrate to dermo-epidermal skin graft in deep burn trauma reduces scarring and need for revision surgeries.

BACKGROUND: [corrected] Deep skin burn injuries, especially those on the face, hands, feet, genitalia and perineum represent significant therapeutic challenges. Autologous dermo-epidermal skin grafts (DESG) have become standard of care for treating deep burns. Additionally, human autologous thrombin activated autologous platelet concentrate (APC) has gained acceptance in the setting of wounds. While each of these interventions has been independently shown to accelerate healing, the combination of the two has never been evaluated. We hypothesized that the addition of platelets (source of growth factors and inhibitors necessary for tissue repair) to the DESG (source of progenitor cells and of tissue proteases necessary for spatial and temporal control of growth regulators released from platelets) would create the optimal environment for the reciprocal interaction of cells within the healing tissues. METHODS: We used clinical examination (digital photography), standardised scales for evaluating pain and scarring, in combination with blood perfusion (laser Doppler imaging), as well as molecular and laboratory analyses. RESULTS: We show for the first time that the combination of APC and DESG leads to earlier relief of pain, and decreased use of analgesics, antipruritics and orthotic devices. Most importantly, this treatment is associated with earlier discharges from hospital and significant cost savings. CONCLUSIONS: Our findings indicate that DESG engraftment is facilitated by the local addition of platelets and by systemic thrombocytosis. This local interaction leads to the physiological revascularization at 1-3 months. We observed significant elevation of circulating platelets in early stages of engraftment (1-7 days), which normalized over the subsequent 7 and 90 days.

Antithrombin-heparin covalent complex reduces microemboli during cardiopulmonary bypass in a pig model.

Transcranial Doppler-detected high-intensity transient signals (HITS) during cardiopulmonary bypass (CPB) surgery have been associated with postoperative neurocognitive dysfunction, suggesting microemboli in the brain could be a contributing factor. HITS occur despite administration of unfractionated heparin (UFH). This study was done to determine whether antithrombin-heparin covalent complex (ATH), a more potent anticoagulant than heparin, can reduce HITS during CPB. In a pig CPB model, ATH, UFH, or UFH + antithrombin (AT) was intravenously administered to female Yorkshire pigs after sternotomy. Twenty minutes later, hypothermic CPB was initiated and continued for 1.25 hours, then normothermia was re-established for 45 minutes. Protamine sulfate was given to neutralize the anticoagulants, and pigs were allowed to recover. HITS were monitored using an arterial flow probe placed over the carotid artery. Compared with UFH (300 or 1000 U/kg), ATH reduced the number of HITS during CPB in a dose-dependent manner. AT (3 mg/kg) + UFH (300 U/kg) resulted in an intermediate HITS rate between UFH and ATH (2 mg/kg in terms of AT). Examination of brain sections for emboli formation confirmed that, similar to HITS, number of thrombi decreased in direct proportion to ATH dosage. These results support the hypotheses that the majority of HITS represent thromboemboli and that ATH reduces emboli formation during CPB.

The role of tumor-and host-related tissue factor pools in oncogene-driven tumor progression.

Oncogenic events play an important role in cancer-related coagulopathy (Trousseau syndrome), angiogenesis and disease progression. This can, in part, be attributed to the up-regulation of tissue factor (TF) and release of TF-containing microvesicles into the pericellular milieu and the circulation. In addition, certain types of host cells (stromal cells, inflammatory cells, activated endothelium) may also express TF. At present, the relative contribution of host- vs tumor-related TF to tumor progression is not known. Our recent studies have indicated that the role of TF in tumor formation is complex and context-dependent. Genetic or pharmacological disruption of TF expression/activity in cancer cells leads to tumor growth inhibition in immunodeficient mice. This occurred even in the case of xenotransplants of human cancer cells, in which TF overexpression is driven by potent oncogenes (K-ras or EGFR). Interestingly, the expression of TF in vivo is not uniform and appears to be influenced by many factors, including the level of oncogenic transformation, tumor microenvironment, adhesion and the coexpression of markers of cancer stem cells (CSCs). Thus, minimally transformed, but tumorigenic embryonic stem (ES) cells were able to form malignant and angiogenic outgrowths in the absence of TF. However, these tumors were growth inhibited in hosts (mice) with dramatically reduced TF expression (low-TF mice). Depletion of host TF also resulted in changes affecting vascular patterning of some, but not all types of tumors. These observations suggest that TF may play different roles growth and angiogenesis of different tumors. Moreover, both tumor cell and host cell compartments may, in some circumstances, contribute to the functional TF pool. We postulate that activation of the coagulation system and TF signaling, may deliver growth-promoting stimuli (e.g. fibrin, thrombin, platelets) to dormant cancer stem cells (CSCs). Functionally, these influences may be tantamount to formation of a provisional (TF-dependent) cancer stem cell niche. As such these changes may contribute to the involvement of CSCs in tumor growth, angiogenesis and metastasis.

Atherosclerosis and vascular aging as modifiers of tumor progression, angiogenesis, and responsiveness to therapy.

It is rarely considered that age-related common vascular co-morbidities may affect therapeutic outcomes of antiangiogenic therapy in cancer. Indeed, the accepted model of human disease consists of 4- to 8-week-old (young) tumor-bearing, but otherwise healthy, experimental mice, yet human cancers are diagnosed and treated in later decades of life when atherosclerosis and vascular diseases are highly prevalent. Here we present evidence that tumor growth and angiogenesis are profoundly altered in mice affected by natural aging and with genetically induced atherosclerosis (in ApoE(-/-) mice). Thus, transplantable tumors (Lewis lung carcinoma and B16F1) grew at higher rates in young (4 to 8 weeks old) ApoE(+/+) and ApoE(-/-) nonatherosclerotic syngeneic recipients than in their old (12 to 18 months old) or atherosclerotic (old/ApoE(-/-)) counterparts. These age-related changes were paralleled by reduced tumor vascularity, lower expression of tumor endothelial marker 1, increased acute tumor hypoxia, depletion of circulating CD45(-)/VEGFR(+) cells, and impaired endothelial sprouting ex vivo. Exposure of tumor-bearing mice to metronomic therapy with cyclophosphamide exerted antimitotic effects on tumors in young hosts, but this effect was reduced in atherosclerotic mice. Collectively, our results suggest that vascular aging and disease may affect tumor progression, angiogenesis, and responses to therapy.

Protein adsorption on polyurethane catheters modified with a novel antithrombin-heparin covalent complex.

Highly anticoagulant covalent antithrombin-heparin complex (ATH) was covalently grafted onto polyurethane catheters to suppress adsorption/activation of procoagulant proteins and enhance adsorption/activation of anticoagulant proteins for blood compatibility. Consistency of catheter coating was demonstrated using immunohistochemical visualization of ATH. The ability of the resulting immobilized ATH heparin chains to bind antithrombin (AT) from plasma, as measured by binding of (125)I-radiolabeled AT, was greater than that for commercially-available heparin-coated catheters, and much greater than for uncoated catheters. Complementary measurements of antifactor Xa (FXa) activity and plasma protein binding were also performed. Both ATH-coated and heparin-coated catheters demonstrated functional binding of exogenous AT. However, the ATH-coated catheters gave a trend towards elevated anti- FXa activities/AT binding ratios, consistent with the higher active pentasaccharide content in starting ATH. Western blot analysis of proteins adsorbed to catheters after incubation with rabbit plasma established protein binding profiles that showed AT and albumin as major plasma proteins adsorbed to ATH-coated catheters, while AT and altered forms of fibrinogen were major plasma protein species adsorbed to heparinized catheters.

Chronic performance of polyurethane catheters covalently coated with ATH complex: a rabbit jugular vein model.

Covalent complexes of antithrombin (AT) and heparin (ATH) have superb anticoagulant activity towards thrombin and factor Xa. Stability of polyurethane central venous catheters covalently modified with radiolabeled ATH was studied using a roller pump with saline or protease P-5147. Saline wash removed loosely bound ATH molecules to decrease graft density from 26 to 12 pmol/cm2. However, only slightly more ATH was removed by strong protease (from 12 to 7 pmol/cm2). To evaluate ATH-coated, heparin-coated, and uncoated catheters, a chronic rabbit jugular vein model was developed with catheters maintained for up to 30-106 days. Lumen occlusion was tested by drawing blood twice daily. Although unmodified or heparin-coated catheters occluded within 5-7 days after insertion, all ATH catheters remained patent throughout the experiment. Scanning electron microscopy (SEM) analysis of heparin and uncoated catheters revealed extensive thrombosis (lumen+mural) while ATH catheters were unaffected. Visual observation showed significant deposition of protein and cells on control and heparin-modified catheters and, to a lesser degree, on ATH-coated surfaces. SEM showed no fibrin inside or outside of ATH catheters, which remained patent in extended studies out to 106 days. Although atomic force microscopy showed ATH coatings to be rough, 6-fold higher anti-factor Xa activity likely contributed to increased patency. Our data confirm that ATH-modified catheters are stable and have superior potency compared to heparin or control catheters.

Biodistribution of covalent antithrombin-heparin complexes.

We have developed a covalent antithrombin-heparin (ATH) complex with advantages compared to non-covalent antithrombin:heparin (AT:H) mixtures. In addition to increased activity, ATH has a longer intravenous half-life that is partly due to reduced plasma protein binding. Given ATH's altered clearance, we investigated biodistribution of ATH in vivo. ATH made from either human plasma-derived AT (pATH) or recombinant human (produced in goats) AT (rhATH) was studied. 125I-ATH + unlabeled carrier was injected into rabbits at different doses. 131I-labeled albumin was administered just before sacrifice as a marker for trapped blood in tissues. Immediately after sacrifice, animal components were removed, weighed, and subsamples were counted for gamma-radioactivity. Percent recoveries of ATH in various organs/compartments at different time points were calculated, and kinetic distribution plots generated. At saturating doses, early disappearance of rhATH from the circulation was much faster than pATH. Co-incident with clearance, 26 +/- 3% of dose for rhATH was liver-associated compared to only 3.7 +/- 0.5% for pATH by 20 min. Also, at early time periods, >60% of all extravascular ATH was liver-associated. Analysis of the vena cava and aorta suggested that vessel wall binding might also account for initial plasma loss of rhATH. By 24 h, most of pATH and rhATH were present as urinary degradation products (51 +/- 3% and 63 +/- 8%, respectively). In summary, systemic elimination of ATH is greatly influenced by the form of AT in the complex, with liver uptake and degradation playing a major role.

Tissue factor in cancer and angiogenesis: the molecular link between genetic tumor progression, tumor neovascularization, and cancer coagulopathy.

Tissue factor (TF), the primary cellular initiator of blood coagulation, is also involved in cancer-related processes such as hypercoagulability (Trousseau syndrome), tumor growth, angiogenesis, and metastasis. Indeed, elevated TF expression by cancer cells and their associated endothelial cells has been reported frequently. Oncogenic events in cancer cells (e.g., expression of mutant K- ras, EGFR, PTEN or p53) lead to an increase in TF levels and activity, and thereby promote tumor aggressiveness, angiogenesis, and hypercoagulability. Like TF, thrombin receptor (protease-activated receptor-1) is also upregulated in cancer cells expressing oncogenic K -ras. Pharmacological antagonists of some of these transforming genes (e.g., epidermal growth factor receptor inhibitors) could diminish TF expression, both locally and systemically, and hence these targeted agents could be viewed as potential indirect and cancer-specific anticoagulants, in addition to their direct antitumor effects. We postulate that levels of circulating TF may be useful in monitoring the biological activity of these agents. Although TF is essential for vascular development, its expression by tumor-associated endothelium appears to play a subtle and seemingly dispensable role. Thus, TF is a pivotal element of the tumor-vascular interface, is involved in many cancer-related processes, and may well constitute a promising new target for anticancer combination therapies in some disease settings.

In vivo rabbit acute model tests of polyurethane catheters coated with a novel antithrombin-heparin covalent complex.

Catheter use has been associated with an increased risk of thrombotic complications. The objective was to make catheters less thrombogenic with the use of antithrombin-heparin covalent complex (ATH). The antithrombotic activity of ATH-coated catheters was compared to uncoated (control) and heparincoated catheters in an acute rabbit model of accelerated occluding clot formation. Anaesthetized rabbits were pre-injected with rabbit (125)I-fibrinogen, followed by insertion of test catheters into the jugular vein. Blood was drawn and held in a syringe, reinjected, then flushed with saline. The experiment was terminated when blood could no longer be withdrawn (occluding clot). Efficacy was defined as the ability of catheters to remain unoccluded. Clot formation, determined by measuring deposition of radiolabeled fibrin, was a secondary endpoint. ATH-coated catheters were resistant to clotting for the full 240-minute duration, while uncoated and heparin-coated catheters had an average clotting time of 78 and 56 minutes, respectively. The patency of ATH coating was dependant on intact heparin pentasaccharide sequences, rather than the chemistries of the basecoat, the PEO spacer arm, or the antithrombin (AT) protein. The ATH coating was stable to ethylene oxide sterilization, modest abrasion, protease attack, and the coating did not appear to leach off the catheter. Surface tension measurements showed that the ATH modified surface was more hydrophilic than uncoated control catheters or heparin-coated catheters. Thus, ATH-coated catheters are better at preventing clots than uncoated or heparin-coated catheters and show promise as an alternative to the currently available catheters in reducing thrombotic complications associated with its use.

Downregulation of the angiogenesis inhibitor thrombospondin 1 in fibroblasts exposed to platelets and their related phospholipids.

Platelets play many important roles in maintenance and formation of blood vessels. The latter is usually attributed to release of direct acting proangiogenic influences, e.g., vascular endothelial growth factor, sphingosine 1 phosphate, and other mediators, though their effects are normally opposed by endogenous angiogenesis inhibitors, including thrombospondin 1 (TSP-1/THSP1). Hence downregulation of TSP-1 is regarded as an important step in the generation of the pro-angiogenic (tumor) stroma. Here we report that platelets induce marked downregulation of TSP-1 (gene transcription and protein) in mouse dermal fibroblasts. This effect is: (i) blocked by inhibitors of sphingosine kinase, (ii) unaffected by inhibitors of G(i)-proteins (pertussis toxin), (iii) recapitulated by sphingosine, (iv) can also be induced by lysophosphatidic acid. These observations suggest a hitherto unappreciated, indirect role of platelets and their phospholipids in angiogenesis, i.e., proangiogenic conditioning of connective tissue stroma through lowering TSP-1 expression.

Investigation of recombinant human elastin polypeptides as non-thrombogenic coatings.

We investigated the use of a recombinant human elastin polypeptide as a coating on synthetic materials with a view to determining if these polypeptides could improve the blood compatibility of cardiovascular devices such as vascular conduits and arterial/venous catheters. Platelet adhesion and activation were studied in vitro using three commercially available synthetic materials: polyethylene terephthalate (Mylar), a poly(tetrafluoroethylene/ethylene) copolymer (Tefzel) and a polycarbonate polyurethane (Corethane). Coated with adsorbed polypeptide, all three synthetic materials demonstrated reduced platelet activation and adhesion in platelet rich plasma in vitro. Compared to non-coated controls, there was a significant decrease (p=0.05) in both platelet microparticle release and P-selectin expression for the polypeptide-coated surfaces. Scanning electron microscopy indicated fewer adhering platelets on coated surfaces compared to non-coated controls. In vivo, in a rabbit model, evaluations of polyurethane catheters coated with the polypeptide showed a marked increase in catheter patency and a significant decrease in fibrin accretion and embolism when compared to uncoated controls. This polypeptide shows a strong potential for use as a non-thrombogenic coating for small diameter vascular grafts. In addition, the results of this study indicate that the elastin polypeptide would be a valuable component of a tissue engineered vascular conduit.

Oncogenes as regulators of tissue factor expression in cancer: implications for tumor angiogenesis and anti-cancer therapy.

Up-regulation of tissue factor (TF) is often observed in cancer. TF is a cell-associated receptor for coagulation factor VII/VIIa, an interaction known to activate the coagulation cascade. At the same time, TF is also known as a mediator of intracellular signaling events that can alter gene expression patterns and cell behavior. Both aspects of TF activity are of possible relevance to tumor growth, metastasis, and angiogenesis, including up-regulation of vascular endothelial growth factor (VEGF). TF up-regulation is often observed on the surfaces of tumor-associated endothelial cells, inflammatory cells, and particularly on cancer cells themselves. In the last case, high TF levels may be associated with poor prognosis and parallel clinical (and genetic) tumor progression. We have proposed elsewhere that TF may be a target of oncogenic events in cancer. Here we discuss our observations suggesting that oncogene-targeting agents may down-regulate TF expression. Such is the effect of treatment with the neutralizing monoclonal antibody (C225) raised against the epidermal growth factor receptor (EGFR) in EGFR-dependent squamous cell carcinoma cells (A431). This two- to threefold TF down-regulation by C225 treatment is paralleled by a decrease in expression of VEGF. It is conceivable that TF participates in signals that regulate VEGF and angiogenesis triggered by activated oncogenic pathways. Therefore, direct targeting of TF in cancer should be considered in combination with other treatment modalities such as oncogene-directed therapies, antiangiogenic agents (e.g., VEGF antagonists), and anti-cancer chemotherapy.

Effect of covalent serpin-heparinoid complexes on plasma thrombin generation on fetal distal lung epithelium.

Extravascular coagulation within the lung airspace is a hallmark of respiratory distress syndrome (RDS) in premature infants. We previously showed that covalent antithrombin-heparin complex (ATH) is superior to noncovalent antithrombin (AT) + heparin (H) mixtures at inhibiting plasma thrombin generation on rat fetal distal lung epithelium (FDLE) in vitro. However, heparin cofactor II (HC) has been shown to selectively inhibit thrombin, which may be advantageous if other enzyme activities are present in the airspace. We compared the abilities of ATH, covalent HC-heparin complex (HCH), and covalent HC-dermatan sulfate (HCD) to inhibit thrombin generation on FDLE in plasmas from either adults or newborns. In the presence of ATH, peak free thrombin generation in adult plasma on the cell surface was reduced by 92% compared with controls (no anticoagulant). However, whereas HCH reduced peak free thrombin generation in adult plasma by 81%, HCD was only able to reduce activity by 33%. All covalent complexes caused a greater decrease in thrombin activity compared with that with the corresponding noncovalent serpin + heparinoid mixtures. Experiments in plasma from newborns resulted in peak free thrombin that was less than or equal to that in adult plasma when covalent conjugates were added. Relative peak free thrombin was proportional to rate of prothrombin consumption and amount of thrombin-inhibitor complexes formed. In vivo, experiments in newborn rats showed that a greater percentage of intratracheally instilled ATH and HCH could be recovered in lung lavage fluid compared withwith that for HCD. In summary, ATH, HCH, and HCD are inhibitors of thrombin generation on FDLE superior to the corresponding noncovalent mixtures, with ATH and HCH being more potent than HCD. Covalent conjugates of AT or HC with H may be preferred in treatment of extravascular coagulation.

Evaluation of DNA aptamers directed to thrombin as potential thrombus imaging agents.

Two DNA aptamers directed against two separate exosites on human alpha-thrombin were evaluated for thrombus-imaging potential. Aptamer ODN 1 is directed to the thrombin substrate binding site (exosite 1). Our finding that ODN 1 competes with fibrin for binding to exosite 1 on thrombin suggests that ODN 1 will not be useful for thrombus imaging. Aptamer ODN 2 is directed against the thrombin heparin binding site (exosite 2). ODN 2 bound to model thrombi that were formed either by clotting purified fibrinogen with thrombin, or by recalcifying citrated plasma. As the thrombin content of thrombi was increased the rate of ODN 2 uptake into preformed thrombi increased, whereas the rate of release of ODN 2 out of preformed thrombi decreased. This in vitro data suggested that ODN 2 might be useful for thrombus imaging because it can bind to exosite 2 on fibrin-bound thrombin. However, in a rabbit jugular vein model using thrombus supplemented with human thrombin, ODN 2 uptake was equal to the ovalbumin control, and did not reflect thrombin content. While the in vitro results with ODN 2 were consistent with thrombus imaging, the rapid clearance of ODN 2 from circulation, combined with slow mass transfer in the clot, seem to work against in vivo thrombin-dependent imaging or washout analysis.

Antithrombin-heparin covalent complex: a possible alternative to heparin for arterial thrombosis prevention.

BACKGROUND: The anticoagulant effect of heparinoids is attributed to their cofactor activity for antithrombin (AT) and heparin cofactor II. In patients with thrombosis, however, thrombin is often protected from AT-dependent, heparin-mediated inactivation. The purpose of this study was to compare the properties of unfractionated/standard heparin (UFH/SH) and those of a novel covalent AT-heparin complex (ATH) in a rabbit arterial thrombosis prevention and bleeding model. METHODS AND RESULTS: Thrombosis in the distal aorta was triggered by vessel wall injury and critical stenosis. Blood flow in the damaged arterial segment was monitored with a flow probe placed distal to the constrictor. Rabbits were given doses of SH (62.5 to 187.5 IU x kg(-1) x 90 min(-1)) or ATH (16 to 65 IU x kg(-1) x 90 min(-1)). Cumulative blood loss from skin incisions was used to assess drug safety. The antithrombotic effects of ATH were greater than those of SH as measured by clot weight, blood flow, and vessel patency; eg, complete thrombus resolution was achieved with ATH (33 to 65 IU/kg), but not SH (125.0 to 187.5 IU/kg). At doses that produced equivalent vessel patency (50% to 60%), blood loss induced by ATH (60.2 microL) was 2.6-fold lower (P<0.05) than that induced by SH (154.6 microL). CONCLUSIONS: In our experimental system, ATH was able to control arterial thrombosis more effectively than its SH precursor, without pronounced bleeding.