Hyperglycemia Potentiates Prothrombotic Effect of Aldosterone in a Rat Arterial Thrombosis Model

We investigated the role of aldosterone (ALDO) in the development of arterial thrombosis in streptozotocin-induced diabetic rats. To evaluate the effect of endogenous ALDO, the rats underwent adrenalectomy (ADX). ADX reduced the development of arterial thrombosis. A 1 h infusion of ALDO (30 μg/kg/h) enhanced thrombosis in adrenalectomized rats, while this effect was potentiated in diabetic rats. ALDO shortened bleeding time, increased plasma levels of tissue factor (TF) and plasminogen activator inhibitor, decreased plasma level of nitric oxide (NO) metabolites, and increased oxidative stress. Moreover, 2 h incubation of human umbilical vein endothelial cells (HUVECs) with ALDO (10−7 M) disrupted hemostatic balance in endothelial cells in normoglycemia (glucose 5.5 mM), and this effect was more pronounced in hyperglycemia (glucose 30 mM). We demonstrated that the acute ALDO infusion enhances arterial thrombosis in rats and hyperglycemia potentiates this prothrombotic effect. The mechanism of ALDO action was partially mediated by mineralocorticoid (MR) and glucocorticoid (GR) receptors and related to impact of the hormone on primary hemostasis, TF-dependent coagulation cascade, fibrinolysis, NO bioavailability, and oxidative stress balance. Our in vitro study confirmed that ALDO induces prothrombotic phenotype in the endothelium, particularly under hyperglycemic conditions.

Coagulation and fibrinolysis in hyperparathyroidism secondary to vitamin D deficiency

Introduction Abnormal coagulation tests have been observed in patients with primary hyperparathyroidism (HPT) suggesting a prothrombotic effect of parathyroid hormone (PTH). Vitamin D deficiency (VIDD) is the most frequent cause of secondary HPT. Aim of our study was to investigate the influence of HPT secondary to moderate-to-severe VIDD and vitamin D replacement on the coagulation and fibrinolysis system. Subjects and methods Prospective cohort study of patients with vitamin D <25 nmol/L with and without HPT, and a control group of patients on vitamin D suppletion. At baseline and after 2 months of vitamin D suppletion (900,000 IU in 2 months), endocrine and coagulation markers were measured. Results 59 patients with VIDD of which 34 had secondary HPT and 36 controls were included. After 2 months of suppletion, vitamin D increased by 399% (VIDD with HPT), 442% (all patients with VIDD) and 6% (controls). PTH decreased by 34% (VIDD with HPT, P < 0.01 for decrease), 32% (all VIDD, P < 0.01) and increased by 8% in the controls (P-values: <0.01 for relative changes between VIDD with HPT or all VIDD patients vs controls). Relative changes in PT, aPTT, fibrinogen, Von Willebrand factor, factors VII, VIII and X, thrombin generation, TAFI, clot-lysis time and d-dimer were not different between patients with VIDD with HPT or all VIDD vs controls. Discussion Secondary HPT due to VIDD does not have a prothrombotic effect. In contrast with previous reports, PTH does not seem to influence coagulation or fibrinolysis, which is relevant because of the high prevalence of VIDD.

Ponatinib and Cardiovascular Complications

Patients with T315I positive CML are resistant to most tyrosine kinase inhibitors (TKIs). Ponatinib (Iclusig) is approved for CML patients with the T315I ABL kinase polymorphism. However, ponatinib treatment is associated with vascular events (myocardial infarction, stroke, coronary artery stenosis, limb ischemia and occlusion, and venous thrombosis) in~29% of patients. The mechanism(s) for these events has not been characterized. We developed a murine model to examine TKIs influence on arterial thrombosis risk. C57BL/6 mice, 18-22 weeks of age and treated with ponatinib by gavage for 14 days at 15 mg/kg PO BID, had significantly shorter carotid artery occlusion times induced by photochemical activation of Rose-Bengal compared to vehicle-treated mice (10.4 ± 2.9 min versus 32.3 ± 4.8 min, p < 0.0001). Mice were treated with ponatinib for 14 days at the 3 mg/kg PO BID, a dose that yields plasma concentrations similar to patients at 45 mg po daily, also had significantly shorter vessel occlusion times compared to control (18.7 ± 3.7 min versus 32.3 ± 4.8 min, p<0.0001). No difference in time to carotid artery occlusion was observed between imatinib at 180 mg/kg PO BID treatment compared to control (32.7 ± 5.6 min versus 32.3 ± 4.8 min, p = 0.85) or nilotinib at 29 mg/kg PO BID treatment compared to vehicle-treated mice (32.8 ± 5.5 min versus 33.8 ± 5.1 min, p = 0.71). These studies show that uniquely ponatinib treatment is prothrombotic. Plasma of ponatinib-treated animals had normal PT, aPTT, and complete blood counts (WBC, RBC, Hgb, Hct, MCV, MCH, MCHC and platelet counts) assays. Also contact activation- and tissue factor-initiated thrombin generation times (TGT) showed no difference between treated and untreated mouse plasma. We next examined the mechanism(s) of ponatinib-induced thrombosis. Ponatinib at 3 mg/kg PO BID daily inhibited p-LynY396 in murine platelets. Lyn is a negative regulator of platelet GPVI. Collagen-related peptide (CRP)-induced expression on murine platelets of the activated heterodimeric complex of α2bβ3 integrins (the JON/A epitope) and the alpha granule constituent P-selectin (CD62) when examined by flow cytometry ex vivo were significantly higher at 3 μg/ml in ponatinib-treated versus untreated mice (p< 0.03). The CRP concentration needed to induce platelet activation in ponatinib-treated mice was significantly lower than untreated mice (p<0.0001, 2-way ANOVA). These data suggested that platelets from ponatinib-treated mice are more GPVI actable. Additional studies with α-thrombin also show ponatinib-treatment makes more active platelets. The threshold for α-thrombin-induced expression of the JON/A epitope also was significantly lower (p<0.0125) at 0.075 and 0.1 nM in ponatinib-treated platelets versus untreated platelets. Likewise, α-thrombin-induced platelet membrane expression of P-selectin also was significantly lower (p<0.025) at 0.075 and 0.1 nM in ex vivo studies of ponatinib-treated platelets. Next, we examined vessel wall for changes in ponatinib-treated mice. Aortic sections showed increased caspase 3 staining in vessel adventitia and surrounding adipose tissue in treated mice, a sign of apoptosis. Also genes involved in vessel anti-thrombosis were altered in 3 mg/kg PO BID ponatinib-treated mice. Expression of mRNA of both COX2 and eNOS and their vasculo-protective transcriptional regulators, Sirt1 and KLF4, respectively, were significantly decreased (p<0.05) in the vessel wall of ponatinib-treated mice. We then sought agents to blunt the prothrombotic changes with ponatinib treatment. Since PPAR-γ agonism elevates Sirt1, and vessel wall Sirt1 is reduced in treated mice, we determined if pioglitazone treatment, a PPAR-γ agonist thiazolidinedione, corrects thrombosis risk after ponatinib in vivo. When ponatinib-treated mice were given oral pioglitazone (10 mg/kg/day po), their short times to thrombosis significantly lengthened (49±6.9 min, p<0.0251) to values like untreated mice. Additionally, neither lisinopril nor atorvastatin ameliorated the ponatinib's prothrombotic effect in vivo. In sum, ponatinib uniquely induces a prothrombotic state due increased platelet activation and reduced vessel wall anti-thrombosis. The effect of ponatinib on platelets may arise in part from its inhibition of p-Lyn. In a murine model of arterial thrombosis, ponatinib's prothrombotic effect is ameliorated by PPAR-γ agonism with pioglitazone. Disclosures No relevant conflicts of interest to declare.

Ginkgo Biloba Extract Blunts the Prothrombotic Effect of Thalidomide on Human Umbilical Endothelial Cells

Patients with multiple myeloma (MM) are at increased risk of developing venous thromboembolism (VTE) compared to general individuals. With the introduction of immunomodulatory drugs (IMiDs), such as thalidomide and lenalidomide, substantially elevates the incidence of VTE. It was reported that patients with MM have significantly increased plasminogen activator inhibitor (PAI-1) level resulting in a decrease of fibrinolytic activity. To prevent VTE in MM patients receiving IMiDs, different prophylactic strategies such as low molecular weight heparin, aspirin and warfarin have been recommended. But most of those recommendations are based on limited evidence. Ginkgo biloba extract (GBE) is one of the most widely used herbal supplements, which has been used in thrombosis prevention. It prompts us to investigate the impact of thalidomide on fibrinolysis and whether GBE has affection on fibrinolysis. In this study, the human umbilical vein endothelial cells (HUVECs) were cultured alone or co-cultured with human multiple myeloma cell line (MMCs) in the medium with or without thalidomide and in the presence or absence of GBE. After 48 hours, culture supernatants were collected and protein concentration of t-PA and PAI-1 were analyzed by enzyme-linked immunosorbent assay. Results showed that MMCs and thalidomide can both altered the profibrinolytic potential of HUVECs by decreasing t-PA and increasing PAI-1 levels. When co-cultured with MMCs, thalidomide can further elevate PAI-1 levels but not further reduce t-PA. Whether HUVECs alone or co-cultured with MMCs, GBE was able to counteract these effects caused by thalidomide through increasing t-PA and decreasing PAI-1 levels. Results in this study indicate that GBE blunts the prothrombotic effect of thalidomide on HUVECs. It supports GBE clinic use for the prevention of VTE induced by IMiDs. Disclosures No relevant conflicts of interest to declare.

Cellular fibronectin containing extra domain A promotes arterial thrombosis in mice through platelet Toll-like receptor 4

Key PointsFn-EDA+ promotes arterial thrombosis. Platelet TLR4 mediates the prothrombotic effect of cellular Fn.

A Novel Role of Protein Disulfide Isomerase in Calpain Regulated Hypoxia Induced Prothrombotic Phenotype

Introduction The increased risk of thromboembolic events under low oxygen environments such as high altitude regions is a well known phenomenon but with little knowledge about molecular events underlying its pathogenesis. We have recently reported the proteomic changes in hyperreactive platelets under hypoxia and demonstrated that increased activity of protease calpain is crucial for the induction of prothrombotic phenotype under hypoxic environment (Tyagi et al, 2014). However, considering the complex nature of hemostatic reactions, multiple aspects of the regulation of hemostatic balance under hypoxia need to be understood. Hypoxic environment creates oxidative stress in biological system which needs to be contained by counteractive factors. One of such factor, the Protein disulfide isomerase (PDI), which catalyzes the formation and rearrangements of disulfide bonds on proteins, has been found to be upregulated in multiple cell types and is believed to be protective under oxidative stress. The extracellular PDI has been recently reported to play a key role in initial thrombotic events in vivo and in vitro, but whether it affects the hemostasis under hypoxia remains unknown. We analyzed the role played by PDI in calpain regulated hypoxia induced prothrombotic phenotype by using specific PDI inhibitor Quercetin-3-rutinoside (Q-3R) in an animal model. Method:The Sprague-Dawley male rats were exposed to simulated hypobaric hypoxia in a specially designed animal decompression chamber maintained at pressure of 366 torr (equivalent to altitude of 6096m) for 3h duration. The exposed group of animals was infused with either Q-3R or vehicle, via tail vein injection prior to hypoxia exposure. The effect of PDI inhibitor on coagulation was analyzed by measuring clotting time and Prothrombin time. The PDI activity was measured in plasma by insulin reduction assay. The plasma calpain activity was measured by fluorescence based assay. The status of oxidative damage was assessed by measuring MDA levels which are hallmark of oxidative stress. We also analyzed the factor V activity in plasma of animals by standard clot based assay using factor V deficient plasma. Results: As previously reported by us, the animals exposed to hypoxic conditions demonstrated prothrombotic tendency as evident in significant shortening of clotting times (PT 84% of control, p < .03) as compared to control animals. The exposed animals preinfused with PDI inhibitors demonstrated further reductions in clotting times. The preinfusion of Q-3R (0.25 to 1.5 mg/kg body wt) significantly shortened the Prothrombin times (71%, p < .01) as compared to respective vehicle control. The hypercoagulative tendency triggered by Q-3R was also evident in drastically reduced activated clotting times in whole blood. Hypoxia induced the activation of PDI which was reflected by significantly elevated activity as compared to control animals. The dose dependent PDI inhibition in Q-3R preinfused animals was confirmed by the fallen activity of PDI in plasma from exposed animals. The calpain activity remained elevated in hypoxic animals (compared to controls) as observed previously. The oxidative damage as measured by MDA levels, was found to be much higher in exposed animals preinfused with Q-3R as compared to the vehicle group. Interestingly, the active factor V was observed to be significantly higher in case of PDI inhibition with largest activity increase in animals infused with highest dose of Q-3R. Conclusion:These results demonstrate the critical role of PDI in regulation of hypoxia induced prothrombotic state. The prevention of increase in activity of PDI in hypoxic animals by using specific PDI inhibitor accelerated the prothrombotic effect of hypoxia. These results appear to be in contrast with recently reported in vivo antithrombotic effect of PDI inhibition. However, as PDI upregulation is considered largely as a protective mechanism, this prothrombotic effect of PDI inhibition under hypoxia seems to be in part due to uncontrolled oxidative stress as shown by higher MDA levels. Also, as factor V activity was shot up by PDI inhibitors, this shows the activation of factor V to be under control of PDI, which can also be explained by role of disulfide linkages in activation of released factor V. Together, these results suggests a novel role of PDI, along with calpain, in regulating hypoxia induced prothrombotic phenotype. Disclosures No relevant conflicts of interest to declare.