Delayed Administration of Nafamostat Mesylate Inhibits Thrombin-Mediated Blood-Spinal Cord Barrier Breakdown During Acute Spinal Cord Injury in Rats

Background: Nafamostat mesylate (NM), an FDA-approved serine protease inhibitor, exerts anti-neuroinflammation and neuroprotective effect on rat spinal cord injury (SCI). However, the time window for NM administration after SCI as well as its underlying mechanism remains unclear. Methods: A series of different first administration time points of NM was tested on rat contusive SCI model. The optimal time window of NM was screened by evaluating hindlimb locomotion and electrophysiology. We performed western blot and immunofluorescence to evaluate the drug target thrombin as well as its downstream Protease activated receptor 1 (PAR-1), and matrix metalloproteinase-9 (MMP9). Enzyme activity assay was used to test thrombin activity. The permeability of blood-spinal cord barrier (BSCB) was assessed by Evans Blue leakage. The infiltration of peripheral inflammatory cell was observed by immunofluorescence.Results: The optimal administration time window of NM was 2-12 h. The thrombin specific inhibitor, Argatroban, had similar pattern. The temporal expression pattern of thrombin peaked at 12 hours and returned to normal level at 7 days post SCI. PAR-1, the thrombin receptor, was observed a significant upregulation after SCI. MMP9, downstream of PAR-1, was also increased along with thrombin and PAR1. The most significant increase of thrombin expression was detected in vascular endothelial cells (ECs). NM significantly downregulated the thrombin and MMP9 expression as well as thrombin activity in the spinal cord, especially in ECs. NM administration at 2-12 h after SCI could inhibit the leakage of Evans blue in the epicenter and upregulate tight junction proteins (TJPs) expression. 8 h administration of NM effectively inhibited the infiltration of peripheral macrophages in the acute SCI. Conclusions: Our study provided preclinical data of NM administration time window in SCI model, which is clinically relevant in the acute SCI. We elucidated the protective mechanism of NM through BSCB protection and anti-neuroinflammation via thrombin intervention.

Risk factors for thromboembolic complications in patients with elevated body mass index during the perioperative period of laparoscopic myomectomy

Background. Uterine fibroid is one of the most common diseases in gynaecological practice accounting from 32 to 70 % according to various authors. In 80 % of cases, fibroids are observed in women of reproductive age. Laparoscopic myomectomy is one of the main methods of surgical treatment for uterine fibroids in wo­men of reproductive age. A large number of advantages of this method of surgical treatment in many cases lead to underestimation of the existing risks such as the risk of thrombosis in women with elevated body mass index (BMI). The purpose of this study is to detect the risk of thrombosis in adequate comprehensive thromboprophylaxis of patients with elevated BMI in the perioperative period of laparoscopic myomectomy using an instrumental method of diagnosis such as low-frequency piezoelectric blood viscometer. Materials and methods. Patients aged 30–45 years undergoing laparoscopic myomectomy (n = 60) were exa­mined. They were divided into 3 groups depending on the BMI and thromboprophylaxis method. Group 1 (n = 16) included women with the BMI < 30 kg/m2, who did not receive thromboprophylaxis. Group 2 (n = 18) consisted of patients with the BMI > 30 kg/m2, who were treated with enoxaparin thromboprophylaxis at a dose of 2,000 anti-Xa IU/0.2 ml subcutaneously (at the BMI of 30–40 kg/m2) and 4,000 anti-Xa IU/0.4 ml subcutaneously (at the BMI > 40 kg/m2). Third group (n = 26) included individuals with the BMI > 30 kg/m2 who received enoxaparin and pentoxifylline for thromboprophylaxis. Results. The low-frequency piezoelectric blood viscometry revealed statistically significant (p < 0.05) deviations from the reference values of the coagulogram towards structural (increased maximum clot density) and chronometric hypercoagulation (acceleration of the time of formation of fibrin-platelet structure T5, shift of the blood clotting time T3 to the left, an increase in the intensity of coagulation drive), elevated thrombin activity (an increase in the constant of thrombin activity, a decrease in T2 time), activation of vascular-platelet hemostasis (reduced period of T1 reaction onset, increased intensity of contact coagulation), inhibition of the lytic activity (a decrease in the intensity of retraction and lysis of the clot) in groups 2 and 3. On the first day after surgery in groups 2 and 3, there was a significant increase (compared to preoperative indicators) in the chronometric indicators such as T1, T2 and T5; a decrease in the structural indicator of maximum clot density, as well as a significant decrease in the constant of thrombin activity, intensity of contact coagulation, which confirms the effectiveness of antithrombotic therapy. However, group 2 reported an increase in the activity of vascular-platelet hemostasis, higher intensity of contact coagulation compared to group 3, shorter T1 and T2 time, and the constant of thrombin activity was higher. On the 5th day after surgery, a normocoagulation trend of hemostatic potential was observed in all groups of patients. Conclusions. The use of low-frequency piezoelectric blood viscometry allows reliably and quickly assessing the kinetics of thrombosis formation.

Thrombin Inhibition Prevents Endothelial Dysfunction and Reverses 20-HETE Overproduction without Affecting Blood Pressure in Angiotensin II-Induced Hypertension in Mice

Angiotensin II (Ang II) induces hypertension and endothelial dysfunction, but the involvement of thrombin in these responses is not clear. Here, we assessed the effects of the inhibition of thrombin activity by dabigatran on Ang II-induced hypertension and endothelial dysfunction in mice with a particular focus on NO- and 20-HETE-dependent pathways. As expected, dabigatran administration significantly delayed thrombin generation (CAT assay) in Ang II-treated hypertensive mice, and interestingly, it prevented endothelial dysfunction development, but it did not affect elevated blood pressure nor excessive aortic wall thickening. Dabigatran’s effects on endothelial function in Ang II-treated mice were evidenced by improved NO-dependent relaxation in the aorta in response to acetylcholine in vivo (MRI measurements) and increased systemic NO bioavailability (NO2− quantification) with a concomitant increased ex vivo production of endothelium-derived NO (EPR analysis). Dabigatran treatment also contributed to the reduction in the endothelial expression of pro-inflammatory vWF and ICAM-1. Interestingly, the fall in systemic NO bioavailability in Ang II-treated mice was associated with increased 20-HETE concentration in plasma (UPLC-MS/MS analysis), which was normalised by dabigatran treatment. Taking together, the inhibition of thrombin activity in Ang II-induced hypertension in mice improves the NO-dependent function of vascular endothelium and normalises the 20-HETE-depedent pathway without affecting the blood pressure and vascular remodelling.

Ischemic stroke in PAR1 KO mice: Decreased brain plasmin and thrombin activity along with decreased infarct volume

Background Ischemic stroke is a common and debilitating disease with limited treatment options. Protease activated receptor 1 (PAR1) is a fundamental cell signaling mediator in the central nervous system (CNS). It can be activated by many proteases including thrombin and plasmin, with various down-stream effects, following brain ischemia. Methods A permanent middle cerebral artery occlusion (PMCAo) model was used in PAR1 KO and WT C57BL/6J male mice. Mice were evaluated for neurological deficits (neurological severity score, NSS), infarct volume (Tetrazolium Chloride, TTC), and for plasmin and thrombin activity in brain slices. Results Significantly low levels of plasmin and thrombin activities were found in PAR1 KO compared to WT (1.6±0.4 vs. 3.2±0.6 ng/μl, p<0.05 and 17.2±1.0 vs. 21.2±1.0 mu/ml, p<0.01, respectively) along with a decreased infarct volume (178.9±14.3, 134.4±13.3 mm3, p<0.05). Conclusions PAR1 KO mice have smaller infarcts, with lower thrombin and plasmin activity levels. These findings may suggest that modulation of PAR1 is a potential target for future pharmacological treatment of ischemic stroke.

Protective role of serpina3c as a novel thrombin inhibitor against atherosclerosis in mice

Abnormal vascular smooth muscle cell (VSMC) proliferation is a critical step in the development of atherosclerosis. Serpina3c is a serine protease inhibitor (serpin) that plays a key role in metabolic diseases. The present study aimed to investigate the role of serpina3c in atherosclerosis and regulation of VSMC proliferation and possible mechanisms. Serpina3c is down-regulated during high-fat diet (HFD)-induced atherosclerosis. An Apoe−/−/serpina3c−/−-double-knockout mouse model was used to determine the role of serpina3c in atherosclerosis after HFD for 12 weeks. Compared with Apoe−/− mice, the Apoe−/−/serpina3c−/− mice developed more severe atherosclerosis, and the number of VSMCs and macrophages in aortic plaques was significantly increased. The present study revealed serpina3c as a novel thrombin inhibitor that suppressed thrombin activity. In circulating plasma, thrombin activity was high in the Apoe−/−/serpina3c−/− mice, compared with Apoe−/− mice. Immunofluorescence staining showed thrombin and serpina3c colocalization in the liver and aortic cusp. In addition, inhibition of thrombin by dabigatran in serpina3c−/− mice reduced neointima lesion formation due to partial carotid artery ligation. Moreover, an in vitro study confirmed that thrombin activity was also decreased by serpina3c protein, supernatant and cell lysate that overexpressed serpina3c. The results of experiments showed that serpina3c negatively regulated VSMC proliferation in culture. The possible mechanism may involve serpina3c inhibition of ERK1/2 and JNK signaling in thrombin/PAR-1 system-mediated VSMC proliferation. Our results highlight a protective role for serpina3c as a novel thrombin inhibitor in the development of atherosclerosis, with serpina3c conferring protection through the thrombin/PAR-1 system to negatively regulate VSMC proliferation through ERK1/2 and JNK signaling.

Thrombin Signaling Contributes to High Glucose-Induced Injury of Human Brain Microvascular Endothelial Cells

Background: Diabetes is one of the strongest disease-related risk factors for Alzheimer’s disease (AD). In diabetics, hyperglycemia-induced microvascular complications are the major cause of end-organ injury, contributing to morbidity and mortality. Microvascular pathology is also an important and early feature of AD. The cerebral microvasculature may be a point of convergence of both diseases. Several lines of evidence also implicate thrombin in AD as well as in diabetes. Objective: Our objective was to investigate the role of thrombin in glucose-induced brain microvascular endothelial injury. Methods: Cultured Human brain microvascular endothelial cells (HBMVECs) were treated with 30 mM glucose±100 nM thrombin and±250 nM Dabigatran or inhibitors of PAR1, p38MAPK, MMP2, or MMP9. Cytotoxicity and thrombin activity assays on supernatants and western blotting for protein expression in lysates were performed. Results: reatment of HBMVECs with 30 mM glucose increased thrombin activity and expression of inflammatory proteins TNFα, IL-6, and MMPs 2 and 9; this elevation was reduced by the thrombin inhibitor dabigatran. Direct treatment of brain endothelial cells with thrombin upregulated p38MAPK and CREB, and induced TNFα, IL6, MMP2, and MMP9 as well as oxidative stress proteins NOX4 and iNOS. Inhibition of thrombin, thrombin receptor PAR1 or p38MAPK decrease expression of inflammatory and oxidative stress proteins, implying that thrombin may play a central role in glucose-induced endothelial injury. Conclusion: Since preventing brain endothelial injury would preserve blood-brain barrier integrity, prevent neuroinflammation, and retain intact functioning of the neurovascular unit, inhibiting thrombin, or its downstream signaling effectors, could be a therapeutic strategy for mitigating diabetes-induced dementia.

Bio-inspired dynamic biomolecule assembling for fine regulation of protein activity

A versatile approach for the fine control of DNA-based hierarchical assembly. Moreover, by a reasonable design of thrombin aptamer structures on the nanoassembly, it can achieve precise regulation of thrombin activity.

Inhibition of thrombin activity by a covalent-binding aptamer and reversal by the complementary strand antidote

We created a DNA-aptamer-type covalent drug where the pharmacological effect can be reversed by the complementary strand antidote.