Combination therapy of normobaric oxygen with hypothermia or ethanol modulates pyruvate dehydrogenase complex in thromboembolic cerebral ischemia.

Pyruvate dehydrogenase complex (PDH) is a brain mitochondrial matrix enzyme. PDH impairment after stroke is particularly devastating given PDH's critical role in the link between anaerobic and aerobic metabolism. This study evaluates the restoration of oxidative metabolism and energy regulation with a therapeutic combination of normobaric oxygen (NBO) plus either therapeutic hypothermia (TH) or ethanol. Sprague-Dawley rats were subjected to middle cerebral artery occlusion with an autologous embolus. One hour after occlusion, tissue-type plasminogen activator (t-PA) was administered alone or with NBO (60%), EtOH (1.0 g/kg), or TH (33°C), either singly or in combination. Neurological deficit score and infarct volume were assessed 24 hr after t-PA-induced reperfusion. PDH activity and reactive oxygen species (ROS) levels were measured 3 and 24 hr after t-PA. Western blotting was used to detect PDH and pyruvate dehydrogenase kinase (PDK) protein expression. After t-PA in ischemic rats, NBO combined with TH or EtOH most effectively decreased infarct volume and neurological deficit. The combined therapies produced greater increases in PDH activity and protein expression as well as greater decreases in PDK expression. Compared with the monotherapeutic approaches, the combined therapies provided the most significant declines in ROS generation. Reperfusion with t-PA followed by 60% NBO improves the efficacy of EtOH or TH in neuroprotection by ameliorating oxidative injury and improving PDH regulation. Comparable neuroprotective effects were found when treating with either EtOH or TH, suggesting a similar mechanism of neuroprotection and the possibility of substituting EtOH for TH in clinical settings. © 2016 Wiley Periodicals, Inc.

Allogeneic stem cell transplantation in paroxysmal nocturnal hemoglobinuria.

BACKGROUND: In the era of eculizumab, identifying patients with paroxysmal nocturnal hemoglobinuria who may benefit from allogeneic stem cell transplantation is challenging. DESIGN AND METHODS: We describe the characteristics and overall survival of 211 patients transplanted for paroxysmal nocturnal hemoglobinuria in 83 EBMT centers from 1978 to 2007. Next, we conducted a comparison with a cohort of 402 non-transplanted patients with paroxysmal nocturnal hemoglobinuria diagnosed between 1950 and 2005 in 92 French centers. We compared the occurrence of complications (i.e. thromboembolism and aplastic anemia) using either an individual or a stratum-matching procedure. RESULTS: After a median follow-up of 5 years, the 5-year overall survival rate ± standard error (%) was 68 ± 3 in the transplanted group (54 ± 7 in the case of thromboembolism, 69 ± 5 in the case of aplastic anemia without thromboembolism and 86 ± 6 in the case of recurrent hemolytic anemia without thromboembolism or aplastic anemia). Only thromboembolism as the indication for transplantation was associated with worse outcome (P=0.03). We identified 24 pairs of transplanted and non-transplanted patients with thromboembolism for the matched comparison, with worse overall survival for the transplanted patients (hazard ratio=10.0; 95% confidence interval, 1.3-78.1; P=0.007). This was confirmed by the global matching procedure (P=0.03). As regards aplastic anemia without thromboembolism, 30 pairs were identified for the matched comparison. It was not observed that transplanted patients had a significantly worse overall survival (hazard ratio=4.0; 95% confidence interval, 0.9-18.9; P=0.06). A global matching procedure was not feasible. CONCLUSIONS: Allogeneic stem cell transplantation is probably not a suitable treatment option for life-threatening thromboembolism in paroxysmal nocturnal hemoglobinuria.

Influence of genetics and non-genetic factors on acenocoumarol maintenance dose requirement in Moroccan patients.

WHAT IS KNOWN AND OBJECTIVE: Coumarin derivatives such as acenocoumarol represent the therapy of choice for the long-term treatment and prevention of thromboembolic diseases. Many genetic, clinical and demographic factors have been shown to influence the anticoagulant dosage. Our aim was to investigate the contribution of genetic and non-genetic factors to variability in response to acenocoumarol in Moroccan patients. METHODS: Our study included 114 adult Moroccan patients, receiving long-term acenocoumarol therapy for various indications. Tests for VKORC1 -1639G>A promoter polymorphism (rs9923231), CYP2C9*2 rs1799853, CYP2C9*3 rs1057910, and CYP4F2 rs2108622 alleles were undertaken using Taq Man(®) Pre-Developed Assay Reagents for allelic discrimination. The statistical analysis was performed using the SAS V9 statistical package. RESULTS AND DISCUSSION: Genotyping showed that the allele frequencies for the SNPs studied were no different to those found in Caucasians population. A significant association was observed between the weekly maintenance dose and the VKORC1 (P = 0·0027) and CYP2C9 variant genotypes (P = 0·0082). A final multivariate regression model that included the target International Normalized Ratio, VKORC1 and CYP2C9 genotypes explained 36·2% of the overall interindividual variability in acenocoumarol dose requirement. WHAT IS NEW AND CONCLUSION: Our study shows large interindividual variability in acenocoumarol maintenance dose requirement in our population. VKORC1 and CYP2C9 variants significantly affected acenocoumarol dose, in-line with results in other populations. For the Moroccan population, the SNPs that have the largest effect on acecoumarol dose are CYP2C9 rs1799853, CYP2C9 rs1057910 and VKORC1 rs9923231.

CYP2C9 and VKORC1 gene polymorphism is inessential for bleeding development under conditions of oral application of anticoagulant acenocoumarol in Russian patients at high risk of thromboembolic complications.

The study included 52 patients at a high risk of thromboembolic complications, with permanent atrial fibrillation. All patients were treated with acenocoumarol for 6 months and the incidence of hemorrhages was evaluated in all of them. All patients were genotyped by CYP2C9 and VKORC1. The presence of CYP2C9*2 and CYP2C9*3 alleles of CYP2C9 locus and AA genotype of VCORC1 gene polymorphic G-1639(3673)A marker was not associated with the development of hemorrhages under conditions of acenocoumarol treatment (p=0.144 for CYP2C9, p=0.809 and 0.918 for VCORC1 in the total group and subgroup of patients with CYP2C9*1/*1 genotype, respectively). The search for other genetic markers of acenocoumarol efficiency and safety is needed for predicting the risk of hemorrhages during this treatment.

Endothelial dysfunction and thromboembolism in children, adolescents, and young adults with acute lymphoblastic leukemia.

Endothelial dysfunction has not previously been investigated as a thrombogenic risk factor among patients with acute lymphoblastic leukemia (ALL), known to be at high risk of thromboembolism. We retrospectively explored the association between three circulating biomarkers of endothelial dysfunction (thrombomodulin, syndecan-1, VEGFR-1) measured in prospectively collected blood samples and risk of thromboembolism in 55 cases and 165 time-matched controls, treated according to the NOPHO ALL2008 protocol. In age-, sex-, and risk group-adjusted analysis, increasing levels of thrombomodulin and VEGFR-1 were independently associated with increased odds of developing thromboembolism (OR 1.37 per 1 ng/mL [95% CI 1.20‒1.56, P < 0.0001] and OR 1.21 per 100 pg/mL [95% CI 1.02‒1.21, P = 0.005], respectively). These associations remained significant when including only samples drawn >30 days before thromboembolic diagnosis. Thrombomodulin levels were on average 3.2 ng/mL (95% CI 2.6-8.2 ng/mL) higher in samples with measurable asparaginase activity (P < 0.0001). Among single nucleotide variants located in or neighboring coding genes for the three biomarkers, none were significantly associated with odds of thromboembolism. If results are validated in another cohort, thrombomodulin and VEGFR-1 could serve as predictive biomarkers, identifying patients in need of preemptive antithrombotic prophylaxis.

Recurrent refractory arterial thromboembolism associated with the Janus kinase 2 V617F mutation.

We report two patients with peripheral vascular disease requiring multiple bilateral radiologic and surgical interventions, and whose disease was unresponsive to conventional anticoagulation and antiplatelet therapy. Although thrombocytosis was only intermittent, analysis of the Janus kinase 2 (JAK2) gene revealed a V617F mutation, thus confirming the presence of an underlying occult myeloproliferative disorder. We propose that JAK2 mutation analysis be considered in patients with recurrent, unexplained arterial events to identify those with occult myeloproliferative disorders.

Absolute risk of venous and arterial thromboembolism in thrombophilic families is not increased by high thrombin-activatable fibrinolysis inhibitor (TAFI) levels.

High levels of thrombin-activatable fibrinolysis inhibitor (TAFI) are a supposed risk factor for thrombosis. However, results from previous studies are conflicting. We assessed the absolute risk of venous and arterial thromboembolism in subjects with high TAFI levels (>126 U/dl) versus subjects with normal levels, and the contribution of other concomitant thrombophilic defects. Relatives from four identical cohort studies in families with either deficiencies of antithrombin, protein C or protein S, prothrombin 20210A, high factor VIII levels, or hyperhomocysteinemia were pooled. Probands were excluded. Of 1,940 relatives, 187 had high TAFI levels. Annual incidences of venous thromboembolism were 0.23% in relatives with high TAFI levels versus 0.26% in relatives with normal TAFI levels (adjusted relative risk [RR] 0.8; 95% confidence interval [CI], 0.5-1.3). For arterial thrombosis these were 0.31% versus 0.23% (adjusted RR 1.4; 95% CI, 0.9-2.2). High levels of factor VIII, IX and XI were observed more frequently in relatives with high TAFI levels. Only high factor VIII levels were associated with an increased risk of venous and arterial thrombosis, independently of TAFI levels. None of these concomitant defects showed interaction with high TAFI levels. High TAFI levels were not associated with an increased risk of venous and arterial thromboembolism in thrombophilic families.

Clinical potential of targeting Bruton's tyrosine kinase.

Targeting Bruton's tyrosine kinase (BTK) with a small-molecule inhibitor may be useful in treatment of BTK-expressing malignancies because of the antiapoptotic function of BTK in cancer cells. Furthermore, BTK inhibitors also exhibit antithrombotic properties, which may be desirable in the context of the increased risk of thromboembolic complications in cancer patients. This review will focus on the role of BTK in drug resistance in cancer, thromboembolism, and various pathologic immune responses, such as graft-versus-host disease. The therapeutic potential of targeting BTK is illustrated by discussion of the biologic activity profile of the rationally designed BTK inhibitor LFM-A13.

Factor VIIa inhibitors: target hopping in the serine protease family using X-ray structure determination.

Selective factor VIIa-tissue factor complex (FVIIa/TF) inhibition is regarded as a promising target for developing new anticoagulant drugs. Compound 1 was discovered from focused screening of serine protease-directed compounds from our internal collection. Using parallel synthesis supported by structure-based drug design, we identified peptidemimetic FVIIa/TF inhibitors (compounds 4-11) containing L-Gln or L-Met as the P2 moiety. However, these compounds lacked the selectivity of other serine proteases in the coagulation cascade, especially thrombin. Further optimization of these compounds was carried out with a focus on the P4 moiety. Among the optimized compounds, 12b-f showed improved selectivity.

Cardiovascular Toxicities with Vascular Endothelial Growth Factor Receptor Tyrosine Kinase Inhibitors in Cancer Patients: A Meta-Analysis of 77 Randomized Controlled Trials.

BACKGROUND AND OBJECTIVE: Use of the vascular endothelial growth factor receptor tyrosine kinase inhibitors (VEGFR-TKIs) has led to considerable improvements in the clinical outcome of patients with various tumor types. However, VEGFR-TKIs may be associated with increased incidence of cardiovascular toxicities. We conducted this meta-analysis to systematically review the risk of cardiovascular toxicities with VEGFR-TKIs in cancer patients. METHODS: The relevant studies of the randomized controlled trials in cancer patients treated with VEGFR-TKIs were retrieved and a systematic evaluation was conducted. EMBASE, MEDLINE, and PubMed were searched for articles published until April 2018. RESULTS: A total of 77 randomized controlled trials and 27,353 patients were included. The current meta-analysis suggests that the use of VEGFR-TKIs significantly increases the risk of developing cardiovascular toxicities, such as all-grade and high-grade hypertension, all-grade bleeding, and all-grade cardiac dysfunction. Hypertension was the most common cardiovascular toxicity. There was no significant increased risk of all-grade and high-grade thromboembolism, high-grade bleeding, and high-grade cardiac dysfunction associated with these agents. CONCLUSIONS: The available data suggest that the use of VEGFR-TKIs is associated with a significantly increased risk of cardiovascular toxicities in cancer patients. Clinicians should be aware of this risk and perform regular cardiovascular monitoring.

Endogenous nitric oxide and prostaglandins synergistically counteract thromboembolism in arterioles but not in venules.

It has been shown that NO and prostacyclin (prostaglandin I(2)) from cultured endothelium synergistically inhibit blood platelet aggregation in vitro. However, it is unknown whether this synergism is also effective in the inhibition of thromboembolism in vivo and, if it is, whether it differs between vessel types. Therefore, the effect of endogenous NO and prostacyclin, in combination or alone, on thromboembolism was studied in an in vivo model. Thromboembolism was induced by micropipette puncture of rabbit mesenteric arterioles and venules (diameter 18 to 40 micrometer). In addition, the influence of wall shear rate was analyzed. In arterioles, the combined inhibition of NO synthase (N(G)-nitro-L-arginine [L-NA] 0.1 mmol/L; local superfusion) and of cyclooxygenase (aspirin [ASA] 100 mg/kg IV) resulted in a pronounced, significant prolongation of embolization duration (median >600 seconds) compared with control (median 153 seconds) or treatment with either L-NA (234 seconds) or ASA (314 seconds). This combined effect of L-NA+ASA was greater than the sum of the individual effects of L-NA and ASA. In contrast, in venules L-NA+ASA had no additional effect on embolization duration (209 seconds) compared with the effect of L-NA alone (230 seconds); ASA alone had no effect (122 seconds; control 72 seconds). Interestingly, only in the L-NA+ASA arterioles did embolization correlate positively with wall shear rate (r(s)=0.687; P=0.028). In conclusion, this study indicates that in arterioles, but not in venules, endogenous NO and prostaglandins synergistically counteract ongoing thromboembolism after vessel wall injury and that the combination of endogenous NO and prostaglandins appears to protect against enhancement of arteriolar thromboembolism by wall shear rate.

Why might statins prevent venous thromboembolism: what needs to be done to know more?

Dyslipidaemia may be a risk factor for venous thromboembolism and 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) may be protective. The current review outlines the cumulative evidence from both the basic and epidemiologic sciences for why this might be so and offers some directions for future research.

Short activated partial thromboplastin times are related to increased thrombin generation and an increased risk for thromboembolism.

This prospective study was designed to investigate whether patients with short activated partial thromboplastin times (aPTTs) have increased thrombin generation and are at increased risk for thromboembolism. During a 4-month period, routine coagulation specimens were screened for the presence of a short or normal aPTT, and, accordingly, 250 specimens were collected. Prothrombin fragment F1 + 2 (F1 + 2) was measured to evaluate thrombin activation, and a second aPTT was performed with a different reagent. Diagnoses were obtained from medical records after conclusion of sample collection. Five to 9 months later, patients were questioned on thromboembolic events during the previous 18 months by questionnaire and telephone interview. F1 + 2 and the incidence of venous thromboses were elevated significantly in the short aPTT group. Unexpectedly, patients with acute bleeding had short aPTTs, but 36% of these also had thromboembolic events during the 18 months proximal to blood collection. These findings were confirmed with the second aPTT reagent. Patients with short aPTTs have increased thrombin generation and are at increased risk for thromboembolism, mainly venous thromboses, despite the fact that a short aPTT can occur in the acute setting of bleeding.

Brain tumors and plasmin inhibitors.

Four different intracranial neoplasms inhibited purified plasmin in an in vitro assay. The greatest inhibition (80%) was observed with extracts of a parasagittal meningioma that had invaded and completely occluded the sinus. Significant inhibition (greater than 40%) was also observed with extracts prepared from a glioma surgically removed from a patient who had suffered three thromboembolic episodes during the preceding several months. Lesser (less than 30%) inhibition was obtained with extracts from two other patients who had no evidence of thromboembolic complications. This report constitutes the first demonstration of plasmin inhibition activity in association with brain tumors. Further studies will be required to test the hypothesis that certain intracranial tumors may escape host antitumor fibrinolytic activity by producing plasmin inhibitors and that plasmin inhibitors may play a role in the thromboembolic complications frequently seen in patients with intracranial neoplasms.

An update on recent patents on thrombin inhibitors (2010 - 2013).

INTRODUCTION: Thromboembolic incidences have increased nearly 33% in the past decade and directly affect nearly 0.5% of the population. Heparin, warfarin and current direct thrombin inhibitors (DTIs), the primary anticoagulants of choice, suffer from several drawbacks. Thus, the search for an antithrombotic devoid of adverse effect continues in earnest. AREAS COVERED: Literature search covering PubMed, SciFinder(™) Scholar, Web of Knowledge, Espacenet, PatentScope and Google Patent Search was used to uncover > 35 patents describing new chemical entities and advances in DTI technologies. Our search uncovered considerable emphasis on the development of larger more complex molecules such as peptide-based inhibitors, prodrug derivatives, bivalent tryptophan zippers, triple action inhibitors and allosteric inhibitors. Advances in formulation technologies for clinically relevant DTIs have also been made. EXPERT OPINION: Thrombin is a multifaceted, dynamic enzyme with both coagulant and anticoagulant functions. Newer DTIs are attempting to fine tune thrombin's activity by targeting allosteric sites or by site-specific targeting of clotting. The complexity of thrombin's functions is driving the design of complex anticoagulants. Advancements in formulations and production processes have attempted to make traditional DTIs more cost effective to produce. The literature reveals a trend to develop a thrombin 'modulator' rather than an 'inhibitor.'

Association between thromboembolic events and the JAK2 V617F mutation in myeloproliferative neoplasms.

Thrombotic complications are a major cause of death in patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), which are closely associated with the JAK2 V617F activating mutation. However, whether the presence of the JAK2 V617F mutation affects thrombotic risk is currently unknown, although some reports have suggested a variable association with thrombosis. Therefore, we investigated the association between JAK2 V617F and various complications, including thrombosis, in Japanese patients with MPNs. We assessed the JAK2 V617F status in 140 patients who were diagnosed or doubted as having some type of MPN by utilizing a JAK2 V617F-specific guanine-quenching probe. JAK2 V617F was detected in 31 of 51 patients (60.8%) with essential thrombocythemia, all 16 patients (100%) with polycythemia vera, 4 of 11 patients (36.4%) with primary myelofibrosis, 2 of 18 patients (11.1%) with other types of MPNs, and none of the 44 patients with doubted MPN. In the 78 patients with classical MPN, JAK2 V617F correlated with a leukocyte count ≥10,000/µl (p=0.046). Complications of thrombosis, hemorrhage, and leukemic transformation occurred in 21 (41.2%), 4 (25.0%), and 3 (27.3%) patients with classical MPN, respectively, and thrombotic events (TE) occurred more frequently in patients with JAK2 V617F than without (p=0.047). Based on these findings, initial screening for the JAK2 mutation and careful monitoring for thrombotic events should be performed in patients with MPN.

The methylenetetrahydrofolate reductase polymorphism (MTHFR c.677C>T) and elevated plasma homocysteine levels in a U.S. pediatric population with incident thromboembolism.

OBJECTIVE: Elevated plasma homocysteine (tHcy) and the MTHFR c.677C>T variant have been postulated to increase the risk of venous thromboembolism (VTE), although mechanisms and implications to pediatrics remain incompletely understood. The objectives of this study were to determine the prevalences of elevated tHcy and MTHFR variant in a pediatric population with VTE or arterial ischemic stroke (AIS), and to determine associations with thrombus outcomes. STUDY DESIGN: Subjects were enrolled in an institution-based prospective cohort of children with VTE or AIS. Inclusion criteria consisted of objectively confirmed thrombus, ≤21years at diagnosis, tHcy measured and MTHFR c.677C>T mutation analysis. Clinical and laboratory data were collected. Frequencies for elevated tHcy and MTHFR variant were compared with NHANES values for healthy US children and also between study groups (VTE vs AIS, provoked vs idiopathic) and by age. RESULTS: The prevalences of hyperhomocysteinemia or MTHFR variant were not increased in comparison to NHANES. tHcy did not differ between those with wild-type MTHFR versus either c.677C>T heterozygotes or homozygotes. There was no association between tHcy or MTHFR variant and thrombus outcomes. CONCLUSION: In this cohort of US children with VTE or AIS, neither the prevalence of hyperhomocysteinemia nor that of MTHFR variant was increased relative to reference values, and adverse thrombus outcomes were not definitively associated with either. While it is important to consider that milder forms of pyridoxine-responsive classical homocystinuria will be detected only by tHcy, we suggest that routine testing of MTHFR c.677C>T genotype as part of a thrombophilia evaluation in children with incident thromboembolism is not warranted until larger studies have been performed in order to establish or refute a link between MTHFR and adverse outcomes.

First report of warfarin dose requirements in patients possessing the CYP2C9*12 allele.

BACKGROUND: Warfarin is the most frequently prescribed anticoagulant in North America and Europe. It is administered as a racemate, but S-warfarin is principally responsible for its anticoagulant activity. Cytochrome P450 (CYP) 2C9 is the enzyme primarily responsible for the metabolism of S-warfarin. Numerous variant alleles of CYP2C9 have been identified. The CYP2C9*12 (rs9332239) allele harbors a P489S substitution in CYP2C9 which has been shown to result in a 40% decline in catalytic activity in vitro. CASES: Four Caucasian patients with a low mean weekly warfarin dose (MWWD) were genotyped for CYP2C9, VKORC1 and APOE variant alleles. None of the four patients carried the common CYP2C9 variant alleles (*2, *3, *5, *6, *7, *8, *9, *11, *13) despite a relatively low MWWD (23.4±7.94 mg) compared to 208 patients carrying the CYP29C9*1 genotype (32.2±12.65 mg). Given that CYP2C9*12 confers decreased in vitro activity to the enzyme, we investigated whether these patients carried this allele. All four patients were CYP2C9*12 CT heterozygotes. Individual comparisons with patients possessing the same VKORC1 and APOE genotypes also demonstrated lower dose requirements in the patients that possessed CYP2C9*12 allele. CONCLUSIONS: There are no reports of the clinical impact of rs9332239 on CYP2C9 substrates. This is the first report of patients with the rare CYP2C9*12 genotype and lower warfarin dose requirements.