Prolylcarboxypeptidase Deficiency Is a Risk Factor for Arterial Thrombosis and Hypertension

Abstract Abstract 651 Background. Prolylcarboxypeptidase (PRCP), an S28 serine protease, degrades bradykinin, angiotensin II, and alpha melanocyte stimulating hormone and activates prekallikrein to plasma kallikrein (Blood 103:4554, 2004). In GWAS, it has been recognized as a risk factor for metabolic syndrome, hypertension, and pre-eclampsia. We postulated that PRCP murine hypomorphs (PRCPgt/gt) have a cardiovascular phenotype. Methods and Results. PRCP is mostly found in kidney in proximal tubules. In arteries, it is found both on endothelium and in media. A gene-trap murine hypomorph was created with 7% mRNA and 23% PRCP antigen in renal tissue. Using the Rose Bengal carotid artery thrombosis models, PRCPgt/gt mice had shorter carotid artery occlusion times (24±3 min [mean±SD]) compared to wild type (52±8 min). On a 4% ferric chloride carotid artery thrombosis assay PRCPgt/gt occluded in 21±8 min whereas wild type do not occlude at 60 min. Pharmacologic inhibition of PRCP with Z-pro-prolinal or plasma kallikrein with soybean trypsin inhibitor, Pro-Phe-Arg-Chloromethylketone or PKSI-572 in 3 mouse strains also shortened the time to carotid artery occlusion. PRCPgt/gt were constitutively hypertensive during the late night cycle (122±5 mm Hg mean arterial pressure vs 114±6 mm Hg for wildtype) as measured by carotid artery telemetry. Treatment of these animals with the mitochondria specific antioxidant mitoTEMPO significantly reduced (113±7 mm Hg) the elevated BP. Plasma angiotensin II and bradykinin levels were unaltered in PRCPgt/gt. PRCPgt/gt plasma had a significant increase in contact activation-induced thrombin generation. Aortic and renal reactive oxygen species (ROS) were increased (3.2-fold and 2.8-fold, respectively) in PRCPgt/gt mice as determined by dihydroethidium (DHE) fluorescence. PRCPgt/gt aortic and renal superoxide measured by lucigenin luminescence also was increased (1.6-fold and 1.7-fold, respectively). In PRCPgt/gt kidneys Amplex Red fluorescence, a measure of hydrogen peroxide, was increased 2.4-fold. Renal tissue had 1.6-fold increased uncoupled eNOS on SDS-PAGE. Arterial occlusion times in PRCPgt/gt were corrected by treatment with antioxidant apocynin or tempol. PRCP siRNA knockdowns in HUVEC or mesenchymal embryonic fibroblasts prepared from PRCPgt/gt embryos had increased constitutive DHE fluorescent ROS (2.1-fold and 1.4-fold, respectively). PRCPgt/gt aorta had decreased expression of Kruppel-like factors 2 and 4, thrombomodulin and eNOS. Moreover, PRCP knockdowns in HUVEC had 36% reduced eNOS mRNA expression. Conclusion. These investigations indicate that PRCP is a specific gene/protein target for arterial thrombosis risk and hypertension. Its presence modulates constitutive cell and tissue ROS. Arterial thrombosis risk is related to effect of ROS on endothelial cell anticoagulant mechanisms. Disclosures: No relevant conflicts of interest to declare.

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The effect of Notrombel on FeCl3-induced carotid artery thrombosis in rats

ZHurnal «Patologicheskaia fiziologiia i eksperimental`naia terapiia» ◽

10.25557/0031-2991.2019.02.50-55 ◽

2019 ◽

pp. 50-55

Author(s):

Е.Ю. Васина ◽

С.Г. Чефу ◽

Н.Н. Петрищев

Keyword(s):

Blood Flow ◽

Carotid Artery ◽

Antioxidant Properties ◽

Artery Occlusion ◽

Carotid Artery Occlusion ◽

Antithrombotic Effect ◽

Platelet Activity ◽

Carotid Artery Thrombosis ◽

Artery Thrombosis ◽

Male Wistar Rats

Цель работы - изучение влияния Нотромбела (ЗАО «Вертекс», Санкт-Петербург, РФ) на развитие экспериментального тромбоза. Методика. Опыты выполнены на крысах-самцах Вистар массой 250-270 г. Нотромбел вводили внутривенно и интрагастрально в течение 10 сут (курсовая доза 25, 50 и 100 мг/кг). В качестве препаратов сравнения использовали ацетилсалициловую кислоту (АСК) и Клопидогрел (курсовая доза 10,5 и 20 мг/кг соответственно). Тромбоз сонной артерии вызывали 60-секундной аппликацией 20% FeCl3. Кровоток регистрировали методом высокочастотной ультразвуковой допплерографии («Минимакс-Допплер-К» Санкт-Петербург, РФ, с рабочей частотой датчика 20 МГц). Прекращение кровотока рассматривали как показатель тромбоокклюзии сонной артерии. Результаты. У контрольных крыс через 20-30 мин после аппликации FeCl3 кровоток в сонной артерии не определялся, что свидетельствовало о развитии тромбоза. Нотромбел как при интрагастральном, так и при внутривенном введении тормозил развитие FeCl3-индуцированного тромбоза сонной артерии. Наиболее эффективной независимо от способа введения оказалась курсовая доза препарата 100 мг/кг, при которой ни в одном из опытов тромбоз не развивался. Антитромботический эффект Нотромбела сопоставим с эффектами АСК и Клопидогрела. В механизме антитромботического влияния Нотромбела имеют значение его прямое ингибирующее влияние на функциональную активность тромбоцитов, а также антиоксидантная активность. Заключение. Нотромбел можно рассматривать как перспективный антитромботический препарат. Aim. To study the effect of Notrombel (ZAO Vertex, St. Petersburg, Russia), a drug of the N, N’-substituted piperazine class containing a carboximidamide group, on experimental thrombosis. Methods. Experiments were carried out on male Wistar rats weighing 250-270 g. Notrombel was administered intravenously and intragastrically for 10 days (course doses 25, 50, and 100 mg/kg). Acetylsalicylic acid (ASA) and Сlopidogrel (course doses 10.5 and 20 mg/kg, respectively) were used as comparator drugs. Carotid artery thrombosis was induced by a 60-s application of 20% FeCl3. Blood flow was recorded by high-frequency ultrasonic dopplerography (Minimax-Doppler-K, St. Petersburg, RF; sensor operating frequency, 20 MHz). Stagnation of blood flow was considered as an indicator of carotid artery occlusion. Results. In control rats 20-30 minutes after the application of FeCl3, the carotid blood flow was undeterminable, which indicated development of thrombosis. Notrombel, both intragastric and intravenous, inhibited the development of FeCl3-induced carotid thrombosis. The most effective dose was 100 mg/kg irrespective of the administration route. Thrombosis has not developed in any experiment. The antithrombotic effect of Notrombel was comparable to that of ASA and Clopidogrel. The Notrombel inhibition of platelet activity and its antioxidant properties contribute to the mechanism of Notrombel antithrombotic effect. Conclusion: Notrombel can be considered as a promising antithrombotic drug.

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Ponatinib and Cardiovascular Complications

Blood ◽

10.1182/blood.v128.22.3055.3055 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3055-3055 ◽

Cited By ~ 1

Author(s):

Alvin H. Schmaier ◽

Alona A. Merkulova ◽

Steven Mitchell ◽

Evi X Stavrou

Keyword(s):

Carotid Artery ◽

Arterial Thrombosis ◽

Vessel Wall ◽

Ex Vivo ◽

Artery Occlusion ◽

Carotid Artery Occlusion ◽

Ppar Γ ◽

Thrombosis Risk ◽

Prothrombotic Effect

Abstract 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.

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Murine prolylcarboxypeptidase depletion induces vascular dysfunction with hypertension and faster arterial thrombosis

Blood ◽

10.1182/blood-2010-11-318527 ◽

2011 ◽

Vol 117 (14) ◽

pp. 3929-3937 ◽

Cited By ~ 63

Author(s):

Gregory N. Adams ◽

Gretchen A. LaRusch ◽

Evi Stavrou ◽

Yihua Zhou ◽

Marvin T. Nieman ◽

...

Keyword(s):

Carotid Artery ◽

Vascular Dysfunction ◽

Artery Occlusion ◽

Carotid Artery Occlusion ◽

Plasma Kallikrein ◽

Cell Dysfunction ◽

Amplex Red ◽

Generation Times ◽

Enos Mrna ◽

Pharmacologic Inhibition

Abstract Prolylcarboxypeptidase (PRCP) activates prekallikrein to plasma kallikrein, leading to bradykinin liberation, and degrades angiotensin II. We now identify PRCP as a regulator of blood vessel homeostasis. β-Galactosidase staining in PRCPgt/gt mice reveals expression in kidney and vasculature. Invasive telemetric monitorings show that PRCPgt/gt mice have significantly elevated blood pressure. PRCPgt/gt mice demonstrate shorter carotid artery occlusion times in 2 models, and their plasmas have increased thrombin generation times. Pharmacologic inhibition of PRCP with Z-Pro-Prolinal or plasma kallikrein with soybean trypsin inhibitor, Pro-Phe-Arg-chloromethylketone or PKSI 527 also shortens carotid artery occlusion times. Aortic and renal tissues have uncoupled eNOS and increased reactive oxygen species (ROS) in PRCPgt/gt mice as detected by dihydroethidium or Amplex Red fluorescence or lucigenin luminescence. The importance of ROS is evidenced by the fact that treatment of PRCPgt/gt mice with antioxidants (mitoTEMPO, apocynin, Tempol) abrogates the hypertensive, prothrombotic phenotype. Mechanistically, our studies reveal that PRCPgt/gt aortas express reduced levels of Kruppel-like factors 2 and 4, thrombomodulin, and eNOS mRNA, suggesting endothelial cell dysfunction. Further, PRCP siRNA treatment of endothelial cells shows increased ROS and uncoupled eNOS and decreased protein C activation because of thrombomodulin inactivation. Collectively, our studies identify PRCP as a novel regulator of vascular ROS and homeostasis.

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Hyperhomocysteinemic Mice Have Increased Susceptibility to Carotid Artery Thrombosis.

Blood ◽

10.1182/blood.v104.11.2616.2616 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2616-2616

Author(s):

Katina M. Wilson ◽

Sanjana Dayal ◽

Teodoro Bottiglieri ◽

Steven R. Lentz

Keyword(s):

Nitric Oxide ◽

Carotid Artery ◽

Control Diet ◽

Green Laser ◽

Wild Type ◽

Carotid Artery Thrombosis ◽

Artery Thrombosis ◽

Total Homocysteine ◽

Oxide Synthase ◽

Endothelial Nitric Oxide

Abstract Hyperhomocysteinemia is a risk factor for cardiovascular disease, stroke, and venous thrombosis. In animal models, hyperhomocysteinemia produces endothelial dysfunction due to decreased bioavailability of endothelium-derived nitric oxide (NO). Because NO has antithrombotic properties, we tested the hypothesis that hyperhomocysteinemia accelerates photochemically-induced carotid artery thrombosis induced in mice. Mice heterozygous for a targeted disruption of the cystathionine β-synthase (Cbs) gene (Cbs+/−) and wild type littermates (Cbs+/+) were fed either a control diet or a high methionine/low folate (HM/LF) diet for 8 months. Plasma total homocysteine was elevated by the HM/LF diet compared with the control diet in both Cbs+/+ (11.6±1.2 vs. 3.7±0.5 μmol/L; p<0.05) and Cbs+/− mice (26.4±3.8 vs. 6.2±0.6 μmol/L; p<0.001). The time to stable occlusion after photochemical injury (rose bengal and green laser) of the carotid artery was 50% shorter in mice fed the HM/HF diet (17.5±2.3 and 18.2±4.0 minutes for Cbs+/+ and Cbs+/− mice, respectively) compared with mice fed the control diet (44.0±9.1 and 31.4±4.7 minutes for Cbs+/+ and Cbs+/− mice, respectively; p<0.001). Using the oxidative dye, dihydroethidium (DHE), increased production of superoxide was detected in Cbs+/+ and Cbs+/− mice fed the HF/HM diet compared with the control diet. To determine whether lack of endothelium-derived NO may accelerate carotid artery thrombosis, mice deficient in endothelial nitric oxide synthase (Nos3−/ −) were also studied. Time to occlusion did not differ between Nos3−/ − mice and wild type mice (55.0±10.2 vs. 42.1±7.1 minutes, p=0.31). We conclude that hyperhomcysteinemia increases susceptibility to experimental thrombosis through a mechanism that is independent of endothelium-derived NO, but may involve oxidative stress.

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LY53857, a 5HT2 Receptor Antagonist, Delays Occlusion and Inhibits Platelet Aggregation in a Rabbit Model of Carotid Artery Occlusion

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646420 ◽

1991 ◽

Vol 66 (03) ◽

pp. 355-360 ◽

Cited By ~ 4

Author(s):

Harve C Wilson ◽

William Coffman ◽

Anne L Killam ◽

Marlene L Cohen

Keyword(s):

Electrical Stimulation ◽

Platelet Aggregation ◽

Carotid Artery ◽

Receptor Antagonist ◽

Artery Occlusion ◽

Carotid Artery Occlusion ◽

Rabbit Platelet ◽

Rabbit Platelets ◽

5Ht2 Receptor

SummaryThe present study was designed to evaluate the effectiveness of the ergoline 5HT2 receptor antagonist, LY53857 in a rabbit model of vascular arterial occlusion. LY53857 (1 and 10 εM) inhibited serotonin amplified platelet aggregation responses to threshold concentrations of ADP in rabbit platelets in vitro. LY53857 (1 εM) not only inhibited the serotonin component of rabbit platelet aggregation, but also inhibited in vitro aggregation induced by ADP (48.7 ± 16.7% inhibition), collagen (76.1 ± 15.9% inhibition) and U46619 (65.2 ± 12.3% inhibition). The effectiveness of this ergoline 5HT2 receptor antagonist in blocking aggregation to ADP, collagen and U46619 may be related to its ability to inhibit a serotonin component of platelet aggregation since rabbit platelets possess high concentrations of serotonin that may be released during aggregation produced by other agents. Based on the effectiveness of LY53857 to inhibit rabbit platelet aggregation, we explored the ability of LY53857 to extend the time to carotid artery occlusion in rabbits following electrical stimulation of the artery. Reproducible carotid artery occlusion was induced in rabbits by moderate stenosis coupled to arterial cross clamping, followed by electrical stimulation. With this procedure, occlusion occurred at 47.0 ± 7 min (n = 30) after initiation of the electrical stimulation. Animals pretreated with LY53857 (50 to 500 εg/kg i.v.) showed a delay in the time to carotid artery occlusion (at 100 εg/kg i.v. occlusion time extended to 164 ± 16 min). Furthermore, ex vivo platelet aggregation from animals treated with LY53857 (300 εg/kg i.v.) resulted in 40.5% inhibition of platelet aggregation in response to the combination of ADP (1 εM) and serotonin (1 εM). These studies document the ability to obtain reproducible arterial occlusion in the rabbit and showed that intravenously administered LY53857 prolonged the time to carotid artery occlusion. Prolongation of carotid artery occlusion time was accompanied by inhibition of serotonin-amplified ADP-induced aggregation in rabbit platelets, an effect observed both in vitro and ex vivo. Thus, the rabbit is a useful model for studying the effectiveness of 5HT2 receptor antagonists in prolonging vascular occlusion induced by insult of the carotid artery.

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