Specific Thrombin Inhibitor Research Articles

Hirudin delays the progression of diabetic kidney disease by inhibiting glomerular endothelial cell migration and abnormal angiogenesis

BackgroundIn the early stages of diabetic kidney disease (DKD), the pathogenesis involves abnormal angiogenesis in the glomerulus. Hirudin, as a natural specific inhibitor of thrombin, has been shown in previous studies to inhibit the migration of various tumor endothelial cells and abnormal angiogenesis. However, its role in DKD remains unclear. MethodsThe effects of hirudin in DKD were studied using spontaneous type 2 diabetic db/db mice (which develop kidney damage at 8 weeks). Network pharmacology was utilized to identify relevant targets. An in vitro high glucose model was established using mouse glomerular endothelial cells (MGECs) to investigate the effects of hirudin on the migration and angiogenic capacity of MGECs. ResultsHirudin can ameliorate kidney damage in db/db mice. Network pharmacology suggests its potential association with the VEGFA/VEGFR2 pathway. Western blot and immunohistochemistry demonstrated elevated protein expression levels of VEGFA, VEGFR2, AQP1, and CD31 in db/db mice, while hirudin treatment reduced their expression. In the MGECs high glucose model, hirudin may reverse the enhanced migration and angiogenic capacity of MGECs in a high glucose environment by altering the expression of VEGFA, VEGFR2, AQP1, and CD31. Moreover, the drug effect gradually increases with higher concentrations of hirudin. ConclusionsThis study suggests that hirudin can improve early-stage diabetic kidney disease kidney damage by inhibiting the migration and angiogenesis of glomerular endothelial cells, thereby further expanding the application scope of hirudin. Additionally, the study found increased expression of AQP1 in DKD, providing a new perspective for further research on the potential pathogenesis of DKD.

Recombinant hirudin suppresses angiogenesis of diffuselarge B-cell lymphoma through regulation of the PAR-1-VEGF.

Hirudin is one of the specific inhibitors of thrombin, which has been confirmed to have strong bioactivities, including inhibiting tumors. However, the function and mechanism of hirudin and protease-activated receptor 1 (PAR-1) in diffuse large B-cell lymphoma (DLBCL) have not been clear. Detecting the expression PAR-1 in DLBCL tissues and cells by RT-qPCR and IHC. Transfected sh-NC, sh-PAR-1, or pcDNA3.1-PAR-1 in DLBCL cells or processed DLBCL cells through added thrombin, Vorapaxar, Recombinant hirudin (RH), or Na2S2O4 and co-culture with EA.hy926. And built DLBCL mice observed tumor growth. Detecting the expression of related genes by RT-qPCR, Western blot, IHC, and immunofluorescence, measured the cellular hypoxia with Hypoxyprobe-1 Kit, and estimated the cell inflammatory factors, proliferation, migration, invasion, and apoptosis by ELISA, CCK-8, flow cytometry, wound-healing and Transwell. Co-immunoprecipitation and pull-down measurement were used to verify the relationship. PAR-1 was highly expressed in DLBCL tissues and cells, especially in SUDHL2. Na2S2O4 induced SUDHL2 hypoxia, and PAR-1 did not influence thrombin-activated hypoxia. PAR-1 could promote SUDHL2 proliferation, migration, and invasion, and it was unrelated to cellular hypoxia. PAR-1 promoted proliferation, migration, and angiogenesis of EA.hy926 or SUDHL2 through up-regulation vascular endothelial growth factor (VEGF). RH inhibited tumor growth, cell proliferation, and migration, promoted apoptosis of DLBCL, and inhibited angiogenesis by down-regulating PAR-1-VEGF. RH inhibits proliferation, migration, and angiogenesis of DLBCL cells by down-regulating PAR-1-VEGF.

Hirudin in the Treatment of Chronic Kidney Disease.

Chronic kidney disease (CKD) is a common public health concern. The global burden of CKD is increasing due to the high morbidity and mortality associated with it, indicating the shortcomings of therapeutic drugs at present. Renal fibrosis is the common pathology of CKD, which is characterized by glomerulosclerosis, renal tubular atrophy, and renal interstitial fibrosis. Natural hirudin is an active ingredient extracted from Hirudo medicinalis, which has been found to be the strongest natural specific inhibitor of thrombin. Evidence based on pharmacological data has shown that hirudin has important protective effects in CKD against diabetic nephrology, nephrotic syndrome, and renal interstitial fibrosis. The mechanisms of hirudin in treating CKD are mainly related to inhibiting the inflammatory response, preventing apoptosis of intrinsic renal cells, and inhibiting the interactions between thrombin and protease-activated receptors. In this review, we summarize the function and beneficial properties of hirudin for the treatment of CKD, and its underlying mechanisms.

Tyrosine phosphorylation of recombinant hirudin increases affinity to thrombin and antithrombotic activity.

Thrombin is one of the key enzymes of the blood coagulation system and a promising target for the development of anticoagulants. One of the most specific natural thrombin inhibitors is hirudin, contained in the salivary glands of medicinal leeches. The medicinal use of recombinant hirudin is limited because of the lack of sulfation on Tyr63, resulting in a 10-fold decrease in activity compared to native (sulfated) hirudin. In the present work, a set of hirudin derivatives was tested for affinity to thrombin: phospho-Tyr63, Tyr63(carboxymethyl)Phe, and Tyr63Glu mutants, which mimic Tyr63 sulfation and Gln65Glu mutant and lysine-succinylated hirudin, which enhance the overall negative charge of hirudin, as well as sulfo-hirudin and desulfo-hirudin as references. Using steered molecular dynamics simulations with subsequent umbrella sampling, phospho-hirudin was shown to exhibit the highest affinity to thrombin among all hirudin analogs, including native sulfo-hirudin; succinylated hirudin was also prospective. Phospho-hirudin exhibited the highest antithrombotic activity in in vitro assay in human plasma. Taking into account the modern methods for obtaining phospho-hirudin and succinylated hirudin, they are prospective as anticoagulants in clinical practice.

Delayed administration of nafamostat mesylate inhibits thrombin-mediated blood–spinal cord barrier breakdown during acute spinal cord injury in rats

BackgroundNafamostat mesylate (nafamostat, NM) is an FDA-approved serine protease inhibitor that exerts anti-neuroinflammation and neuroprotective effects following rat spinal cord injury (SCI). However, clinical translation of nafamostat has been limited by an unclear administration time window and mechanism of action.MethodsTime to first dose of nafamostat administration was tested on rats after contusive SCI. The optimal time window of nafamostat was screened by evaluating hindlimb locomotion and electrophysiology. As nafamostat is a serine protease inhibitor known to target thrombin, we used argatroban (Arg), a thrombin-specific inhibitor, as a positive control in the time window experiments. Western blot and immunofluorescence of thrombin expression level and its enzymatic activity were assayed at different time points, as well its receptor, the protease activated receptor 1 (PAR1) and downstream protein matrix metalloproteinase-9 (MMP9). Blood–spinal cord barrier (BSCB) permeability leakage indicator Evans Blue and fibrinogen were analyzed along these time points. The infiltration of peripheral inflammatory cell was observed by immunofluorescence.ResultsThe optimal administration time window of nafamostat was 2–12 h post-injury. Argatroban, the thrombin-specific inhibitor, had a similar pattern. Thrombin expression peaked at 12 h and returned to normal level at 7 days post-SCI. PAR1, the thrombin receptor, and MMP9 were significantly upregulated after SCI. The most significant increase of thrombin expression was detected in vascular endothelial cells (ECs). Nafamostat and argatroban significantly downregulated thrombin and MMP9 expression as well as thrombin activity in the spinal cord. Nafamostat inhibited thrombin enrichment in endothelial cells. Nafamostat administration at 2–12 h after SCI inhibited the leakage of Evans Blue in the epicenter and upregulated tight junction proteins (TJPs) expression. Nafamostat administration 8 h post-SCI effectively inhibited the infiltration of peripheral macrophages and neutrophils to the injury site.ConclusionsOur study provides preclinical information of nafamostat about the administration time window of 2–12 h post-injury in contusive SCI. We revealed that nafamostat functions through inhibiting the thrombin-mediated BSCB breakdown and subsequent peripheral immune cells infiltration.

Modulation of the Thrombin Pathway Restores LTP in a Pilocarpine Mice Model of Status Epilepticus

BackgroundStatus epilepticus (SE) leads to memory impairment following a seizure, attributed to long-term potentiation (LTP) reduction. Thrombin, a coagulation factor that activates protease-activated receptor 1 (PAR1) is involved in cognitive impairment following traumatic brain injury by reducing hippocampal LTP and in seizures as seen in a SE pilocarpine-induced mice model. Thrombin pathway inhibition prevents this cognitive impairment. We evaluated the effect of thrombin pathway inhibition in the pilocarpine-induced SE mice model, on LTP, hippocampal, and serum markers for inflammation, the PAR1 pathway, and neuronal cell damage.MethodsSE was induced by injecting C57BL/6J mice with pilocarpine. Before pilocarpine injection, mice were injected with either the specific thrombin inhibitor α-NAPAP [Nα-(2-naphthalene-sulfonylglycyl)-4-amidino-DL-phenylalaninepiperidide], the PAR1 antagonist SCH79797, or vehicle-only solution. Recordings of excitatory postsynaptic potentials (EPSP) were conducted from hippocampal slices 24 h following pilocarpine injection. Hippocampal real-time PCR for the quantification of the PAR1, prothrombin, and tumor necrosis factor α (TNF-α) mRNA expression levels was conducted. Serum levels of neurofilament light chain (NfL) and TNF-α were measured by a single molecule array assay.ResultsThe EPSP was reduced in the pilocarpine-induced SE mice (p < 0.001). This reduction was prevented by both NAPAP and SCH79797 treatments (p < 0.001 for both treatments). Hippocampal expression of TNF-α was elevated in the pilocarpine-induced SE group compared to the control (p < 0.01), however, serum levels of TNF-α were not changed. NfL levels were elevated in the pilocarpine-induced SE group (p = 0.04) but not in the treated groups.ConclusionsPilocarpine-induced SE reduces LTP, in a thrombin PAR1-related mechanism. Elevation of serum NfL supports neuronal damage accompanying this functional abnormality which may be prevented by PAR1 pathway modulation.

Resolving Interference of Direct Oral Anticoagulants (DOACs) on Routine Coagulation, Thrombophilia, and Lupus Anticoagulant Testing Using Activated Carbon

Background: Direct (anti-IIa and anti-Xa) oral anticoagulants (DOACs) have a significant impact on various coagulation test results. As the correct interpretation of these tests is mandatory to prevent misclassification and subsequent clinical consequences, withholding treatment could be necessary, with the associated risk of thrombosis.Aims: To evaluate the performance of the activated carbon DOAC-Remove (5-Diagnostics, Basel, Switzerland) in extracting DOACs from plasma samples and its effect on various routine and esoteric coagulation test results.Patients, Materials and Methods: Left-over plasmas from patients treated or not with DOAC obtained in the routine laboratory workload were evaluated. Briefly, 0.8 mL of plasma sample was incubated with 1 tablet of the activated carbon for 10 min at room temperature using a rotating shaker. After a 2 min-centrifugation at 2000x g and room temperature, the supernatant was pipetted before being analyzed. Tests were performed before and after such incubation. DOACs were measured using either a specific direct thrombin inhibitor or an anti-Xa assay with specific calibrations. Routine coagulation tests (PT, aPTT, and factor(F)V, FVIII:C, FIX, fibrinogen, and D-dimer) were performed, as well as thrombophilia panel [antithrombin, protein C (PC, chromogenic and clotting assays), PS (free PS antigen and clotting assays)], and lupus anticoagulant (LA) panel [silica clotting time (SCT), and dilute Russell venom clotting time (dRVVT)]. All assays were performed using reagents from Werfen (Bedford, MA, USA) on the ACL TOP 700 analyzer. As the distribution of data was not normal, test results were compared using non-parametric tests.Results: We evaluated a total of 756 plasma samples, obtained from patients treated with dabigatran (n=139, median concentration 129 ng/mL [range:18-905]), rivaroxaban (n=157, median concentration 159 ng/mL [range:19-815]), or apixaban (n=155, median concentration=154 ng/mL (range:11-510), and from 305 patients with various disease states and not on DOAC including 35 patients on coumadin and 32 patients on heparin (UFH: n=18 ; LMWH: n=14).In untreated patients, the DOAC-Remove had no significant impact on test results (n>30 for each parameter), except for D-dimer, free PS and FVIII:C. However, the mean biases, evaluated according to Bland-Altman, were below the accepted limits, and changes would not have had any clinical relevance.In the plasma from patients treated on DOAC, the DOAC-Remove eliminated all the three DOACs, with levels far below the detection limit of the techniques after a 10 min-incubation, leading to a dramatic correction of the DOAC-induced prolongations of PT and APTT, as well as of their lowering effect on FV, FVIII and FIX activities. The same applied to the DOAC-induced elevation of PC and PS anticoagulant activities (clotting assays), and antithrombin activity in the plasma from patients on rivaroxaban and apixaban.DRVVT screen/confirm ratios, which were above the normal ranges in 45% of the patients on dabigatran (n=31), and 78% of the patients on rivaroxaban (n=32), were normalized in most samples after a 10 min-incubation with the carbon and remained positive in only 10% of the patients on dabigatran and 12% of the patients on rivaroxaban. The impact of the DOACs on the SCT screen/confirm ratio was less noticeable with baseline positive test results in only one of the samples from patients on dabigatran (n=31), and in 2 patients on rivaroxaban (n=32). Treatment with the carbon leaded to a correction in the positive sample from patient on dabigatran and in one of the two positive samples from patients on rivaroxaban. Both dRVVT and SCT test results were within the normal range in the plasma from 32 patients on apixaban before and after treatment with the activated carbon.As expected, fibrinogen, PC activity evaluated using a chromogenic assay, free PS antigen concentration, VWF:RCo and VWF:Ag were not affected by any of the 3 DOACs, and test results were not significantly different before and after incubation with the activated carbon.Conclusion: The DOAC-Remove had no effect on test results obtained in the plasma from untreated patients and from patients on traditional anticoagulants. It effectively removed all 3 tested DOACs from the plasma of treated patients, allowing an accurate measurement of routine and esoteric coagulation tests in DOAC treated patients without withholding the treatment. DisclosuresNo relevant conflicts of interest to declare.

Effect of hirudin on arterialized venous flap survival in rabbits

Arterialized venous flap (AVF) is limited in clinical application because its survival remains inconsistent and its exact survival mechanism is still unclear. Hirudin is an effective thrombin specific inhibitor, which is isolated from the salivary gland secretions of the leech. Our study evaluated the impact of hirudin on the viability of AVFs in rabbits. Thirty-six rabbits were randomly divided into three groups: sham group (physiological perfusion), control group (AVF), and hirudin group (AVF + hirudin). In hirudin group, 20 antithrombin units (ATU) hirudin (2.5 ml) were injected into each flap. In sham group and control group, the same amount of normal saline was injected into each flap. Status of flap survival, water content, vascular perfusion, histopathology, expression of CD34, VEGF, eNOS and HIF-1α were analyzed in each group. Analysis of oxidative stress was performed by measuring the activity of superoxide dismutase (SOD) and malondialdehyde (MDA). Compared with flaps in sham group with physiological perfusion mode, results of survival rate, perfusion status, SOD activity, expression of CD34, VEGF, and eNOS of AVFs in control group were significantly lower, while water content, MDA level and expression of HIF-1α were higher. The flap condition of AVFs injected with hirudin in hirudin group was improved significantly, and the results were similar to sham group. Our findings revealed that hirudin can effectively improve survival of AVF.

Pharmacological Activities and Mechanisms of Hirudin and Its Derivatives - A Review.

Hirudin, an acidic polypeptide secreted by the salivary glands of Hirudo medicinalis (also known as “Shuizhi” in traditional Chinese medicine), is the strongest natural specific inhibitor of thrombin found so far. Hirudin has been demonstrated to possess potent anti-thrombotic effect in previous studies. Recently, increasing researches have focused on the anti-thrombotic activity of the derivatives of hirudin, mainly because these derivatives have stronger antithrombotic activity and lower bleeding risk. Additionally, various bioactivities of hirudin have been reported as well, including wound repair effect, anti-fibrosis effect, effect on diabetic complications, anti-tumor effect, anti-hyperuricemia effect, effect on cerebral hemorrhage, and others. Therefore, by collecting and summarizing publications from the recent two decades, the pharmacological activities, pharmacokinetics, novel preparations and derivatives, as well as toxicity of hirudin were systematically reviewed in this paper. In addition, the clinical application, the underlying mechanisms of pharmacological effects, the dose-effect relationship, and the development potential in new drug research of hirudin were discussed on the purpose of providing new ideas for application of hirudin in treating related diseases.

Markers for neural degeneration and regeneration: novel highly sensitive methods for the measurement of thrombin and activated protein C in human cerebrospinal fluid.

Inflammation and coagulation are tightly interconnected in the pathophysiology of neuronal diseases. Thrombin, a pro-coagulant serine protease is associated with neurodegeneration and its indirect inhibitor, activated protein C (aPC), is considered neuroprotective. While levels of thrombin and aPC activity are readily measured in the blood, similar assays in the cerebrospinal fluid (CSF) have not been described. The aim of this study was to establish a specific and sensitive enzymatic assay to measure both thrombin and aPC activity in the CSF. CSF was collected from 14 patients with suspected normal pressure hydrocephalus served as a control group, while seven patients with central nervous system infections served as an acute neuro-inflammatory study group and one sample of CSF following traumatic lumbar puncture served as a positive control. Thrombin and aPC activities were measured by fluorescence released by specific proteolytic cleavage in the presence of endopeptidase and amino-peptidase inhibitors to ensure specificity. Specificity of the method was verified by thrombin and serine-protease inhibitors N-alpha-((2-naphthylsulfinyl)glycyl)-DL-p-amidinophenylalanylpiperidine and phenylmethanesulfonyl fluoride. Inhibition of thrombin activity by CSF samples and levels of specific thrombin inhibitors were also assessed. Thrombin and aPC activities were reliably measured and were significantly higher in the CSF of patients with central nervous system infections compared to normal pressure hydrocephalus controls, suggesting the involvement of these factors in neuro-inflammation. CSF thrombin activity levels in the presence of known thrombin concentration were high in patients with central nervous system infections, and low in normal pressure hydrocephalus patients. Quantification of endogenous thrombin inhibitors protease nexin 1, amyloid precursor protein and anti-thrombin III in CSF by western blot indicated a significant elevation of amyloid precursor protein in infectious CSF. In conclusion, this study describes a novel and sensitive assay aimed at the detection of thrombin and aPC activity in CSF. This method may be useful for measuring these factors that reflect degenerative and protective influences of coagulation on neurological disorders. The study procedure was approved by the Ethics Committee of the Chaim Sheba Medical Center (approval No. 4245-17-SMC) on October 18, 2018.

Dabigatran Suppresses PAR-1/SphK/S1P Activation of Astrocytes in Experimental Autoimmune Encephalomyelitis Model.

Multiple sclerosis (MS) is an inflammatory autoimmune disease affecting the central nervous system (CNS) that currently does not have any effective treatment. Experimental autoimmune encephalomyelitis (EAE) is often employed as a model to mimic the clinical manifestations of MS, mainly CNS demyelination. Coagulation is known to participate in crosstalk with inflammation and autoimmunity. We herein explored the correlation between the coagulation cascade and CNS immune diseases in vitro using primary astrocytes isolated from mice and in vivo using a mouse model of EAE. We showed that dabigatran, a clinical oral anti-coagulant drug, suppressed the thrombin-induced activation of astrocytes, and the underlying mechanisms are related to the activity of protease-activated receptor-1 (PAR-1), sphingosine-1-phosphate (S1P), and sphingosine kinases (SphKs). Importantly, dabigatran effectively recovered neurological function, reduced inflammation in the spinal cord, and prevented spinal cord demyelination caused by EAE. We suggest that dabigatran, a specific inhibitor of thrombin, antagonized the effect of thrombin in astrocytes by limiting the activation of PAR-1, in turn downregulating SphK1 and disrupting S1P receptor signaling. These findings reveal critical information about the relationship between coagulation mechanisms and CNS immune diseases and will contribute to the clinical translation and development of therapeutic strategies against MS.

The role of thrombin in the pathogenesis of diabetic neuropathy.

Diabetic neuropathy is common and disabling despite glycemic control. Novel neuroprotective approaches are needed. Thrombin and hypercoagulability are associated with diabetes and nerve conduction dysfunction. Our aim was to study the role of thrombin in diabetic neuropathy. We measured thrombin activity by a biochemical assay in streptozotocin (STZ)-induced diabetic neuropathy in male Sprague-Dawley rats. Neuropathy severity was assessed by thermal latency and nerve conduction measures. Thermal latencies were longer in diabetic rats, and improved with the non-specific serine-protease inhibitor Tosyl-L-lysine-chloromethyl ketone (TLCK) treatment (p<0.01). The tail nerve of diabetic rats showed slow conduction velocity (p˂0.01), and interestingly, increased thrombin activity was noted in the sciatic nerve (p˂0.001). Sciatic nodes of Ranvier and the thrombin receptor, protease activated receptor 1 (PAR1) reactivity showed abnormal morphology in diabetic animals by immunofluorescence staining (p<0.0001). Treatment of diabetic animals with either the specific thrombin inhibitor, N-alpha 2 naphtalenesulfonylglycyl alpha-4 amidino-phenylalaninepiperidide (NAPAP) or TLCK preserved normal conduction velocity, (p˂0.01 and p = 0.01 respectively), and prevented disruption of morphology (p˂0.05 and p˂0.03). The results establish for the first time an association between diabetic neuropathy and excessive activation of the thrombin pathway. Treatment of diabetic animals with thrombin inhibitors ameliorates both biochemical, structural and electrophysiological deficits. The thrombin pathway inhibition may be a novel neuroprotective therapeutic target in the diabetic neuropathy pathology.

Blocking Thrombin Significantly Ameliorates Experimental Autoimmune Neuritis.

Thrombin and its protease-activated receptor 1 (PAR1) are potentially important in peripheral nerve inflammatory diseases. We studied the role of thrombin and PAR1 in rat experimental autoimmune neuritis (EAN), a model of the human Guillain-Barré syndrome (GBS). EAN was induced by bovine peripheral myelin with complete Freund's adjuvant (CFA). Thrombin activity in the sciatic nerves, clinical scores and rotarod performance were measured. Thrombin activity in the sciatic nerve was elevated in EAN compared to CFA control rats (sham rats) (p ≤ 0.004). The effect of blocking the thrombin-PAR1 pathway was studied using the non-selective thrombin inhibitor N-Tosyl-Lys-chloromethylketone (TLCK), and the highly specific thrombin inhibitor N-alpha 2 naphtalenesulfonylglycyl 4 amidino-phenylalaninepiperidide (NAPAP). In-vitro TLCK and NAPAP significantly inhibited specific thrombin activity in EAN rats sciatics (p<0.0001 for both inhibitors). Treatment with TLCK 4.4 mg/kg and NAPAP 69.8 mg/kg significantly improved clinical and rotarod scores starting at day 12 and 13 post immunization (DPI12, DPI13) respectively (p < 0.0001) compared to the untreated EAN rats. In nerve conduction studies, distal amplitude was significantly lower in EAN compared to sham rats (0.76 ± 0.34 vs. 9.8 ± 1.2, mV, p < 0.0001). Nerve conduction velocity was impaired in EAN rats (23.6 ± 2.6 vs. sham 43 ± 4.5, m/s p = 0.01) and was normalized by TLCK (41.2 ± 7.6 m/s, p < 0.05). PAR1 histology of the sciatic node of Ranvier indicated significant structural damage in the EAN rats which was prevented by TLCK treatment. These results suggest the thrombin-PAR1 pathway as a possible target for future intervention in GBS.

Hirudin ameliorates immunoglobulin A nephropathy by inhibition of fibrosis and inflammatory response

Immunoglobulin A nephropathy (IgAN) is characterized by mesangial IgA and IgG co-deposition. As the clinical course of IgAN is highly variable, a lot of patients will eventually develop to end-stage renal disease (ESRD) within years. Hirudin, a potent and specific thrombin inhibitor, has been reported to treat IgAN with hematuria, but the mechanism is unclear. Our study aims to explore the potential of hirudin and the underlying mechanism in the treatment of IgAN. The establishment of IgAN model was set up in rats through oral and intravenous immunization with bovine gamma-globulin (BGG). Results suggested that hirudin could reduce the increased level of proteinuria, serum creatinine and urea nitrogen in IgAN models. Besides that, hirudin ameliorated the elevated number of apoptotic bodies and expressions of apoptosis-related proteins (caspase-3 and caspase-9) in IgAN model. The fibrosis indexes (transforming growth factor β-1 (TGF-β1), Collagen-IV (CoI-IV) and Fibronectin-1) of kidney were remarkably suppressed in IgAN rats treated with hirudin compared with IgAN rats with no further treatment. IgAN rats exhibited remarkably increased inflammatory factors (IL-1β, IL-6, and IL-18), while hirudin treatment significantly alleviated these alterations. Moreover, the reduced levels of CD4+CD25+Foxp3+ Treg and CD4+IFN-γ+ Th1/CD4+IL-4+ Th2 could be reversed by hirudin in IgAN model. Furthermore, in the process of IgAN, hirudin could inactivate various pathways (IκBα, NF-κB, TNF-α, and VCAM-1) compared with IgAN model group. Taken together, our study indicated that hirudin could ameliorate IgAN through suppressing fibrosis and inflammatory response. These findings provide a new therapeutic method to treat IgAN.

Thrombin-Mediated Degradation of Human Cardiac Troponin T.

Cardiac troponin T (cTnT) is an acknowledged biomarker of acute myocardial infarction (AMI) that is known to be prone to proteolytic degradation in serum. Such degradation is usually explained by the action of μ-calpain, although there could be other candidates for that role. In the current study, we explored the hypothesis that thrombin-mediated cTnT cleavage occurs as a result of the serum sample preparation. cTnT degradation was studied by using immunoblotting and mass spectrometry (MS) analysis. The comparison of cTnT isolated from AMI heparin plasma and serum samples showed that cTnT in the plasma samples was mainly present as the full-sized molecule (approximately 35 kDa), while in serum samples it was present as a 29-kDa fragment. The incubation of recombinant cTnT, or native ternary cardiac troponin complex with thrombin or in normal human serum (NHS), resulted in the formation of a 29-kDa product that was similar to that detected in AMI serum samples. No cTnT degradation was observed when thrombin or NHS was pretreated with hirudin, a specific inhibitor of thrombin, or during incubation of troponin in normal heparin plasma. When the products of thrombin-mediated cTnT proteolysis were analyzed by MS, 2 fragments consisting of amino acid residues (aar) 2-68 and 69-288 were identified, which suggests that thrombin cleaves cTnT between R68 and S69. The results of this study suggest that the 29-kDa fragment of cTnT in AMI serum samples mainly appears due to the cleavage by thrombin during serum sample preparation.

Revisiting antithrombotic therapeutics; sculptin, a novel specific, competitive, reversible, scissile and tight binding inhibitor of thrombin

Thrombin is a multifunctional enzyme with a key role in the coagulation cascade. Its functional modulation can culminate into normal blood coagulation or thrombosis. Thus, the identification of novel potent inhibitors of thrombin are of immense importance. Sculptin is the first specific thrombin inhibitor identified in the transcriptomics analysis of tick’s salivary glands. It consists of 168 residues having four similar repeats and evolutionary diverged from hirudin. Sculptin is a competitive, specific and reversible inhibitor of thrombin with a Ki of 18.3 ± 1.9 pM (kon 4.04 ± 0.03 × 107 M−1 s−1 and koff 0.65 ± 0.04 × 10−3 s−1). It is slowly consumed by thrombin eventually losing its activity. Contrary, sculptin is hydrolyzed by factor Xa and each polypeptide fragment is able to inhibit thrombin independently. A single domain of sculptin alone retains ~45% of inhibitory activity, which could bind thrombin in a bivalent fashion. The formation of a small turn/helical-like structure by active site binding residues of sculptin might have made it a more potent thrombin inhibitor. In addition, sculptin prolongs global coagulation parameters. In conclusion, sculptin and its independent domain(s) have strong potential to become novel antithrombotic therapeutics.

Thrombin-Induced Podocyte Injury Is Protease-Activated Receptor Dependent.

Nephrotic syndrome is characterized by massive proteinuria and injury of specialized glomerular epithelial cells called podocytes. Studies have shown that, whereas low-concentration thrombin may be cytoprotective, higher thrombin concentrations may contribute to podocyte injury. We and others have demonstrated that ex vivo plasma thrombin generation is enhanced during nephrosis, suggesting that thrombin may contribute to nephrotic progression. Moreover, nonspecific thrombin inhibition has been shown to decrease proteinuria in nephrotic animal models. We thus hypothesized that thrombin contributes to podocyte injury in a protease-activated receptor-specific manner during nephrosis. Here, we show that specific inhibition of thrombin with hirudin reduced proteinuria in two rat nephrosis models, and thrombin colocalized with a podocyte-specific marker in rat glomeruli. Furthermore, flow cytometry immunophenotyping revealed that rat podocytes express the protease-activated receptor family of coagulation receptors in vivo High-concentration thrombin directly injured conditionally immortalized human and rat podocytes. Using receptor-blocking antibodies and activation peptides, we determined that thrombin-mediated injury depended upon interactions between protease-activated receptor 3 and protease-activated receptor 4 in human podocytes, and between protease-activated receptor 1 and protease-activated receptor 4 in rat podocytes. Proximity ligation and coimmunoprecipitation assays confirmed thrombin-dependent interactions between human protease-activated receptor 3 and protease-activated receptor 4, and between rat protease-activated receptor 1 and protease-activated receptor 4 in cultured podocytes. Collectively, these data implicate thrombinuria as a contributor to podocyte injury during nephrosis, and suggest that thrombin and/or podocyte-expressed thrombin receptors may be novel therapeutic targets for nephrotic syndrome.

Thrombin contributes to protective immunity in pneumonia‐derived sepsis via fibrin polymerization and platelet–neutrophil interactions

Background Innate immunity and coagulation are closely linked during sepsis. Their interaction can be detrimental to the outcome because of microvascular failure but can also enhance host defense. The role of thrombin therein has not been fully elucidated. Objective We aimed to investigate the contribution of thrombin to the host response during pneumonia-derived sepsis. Methods Mice treated with the specific thrombin inhibitor dabigatran or control chow were infected with the common human sepsis pathogen Klebsiella (K.) pneumoniae via the airways. In subsequent infection experiments, mice were additionally treated with ancrod to deplete fibrinogen. Ex vivo Klebsiella growth was assessed by incubating human whole blood or specific blood components in various conditions with Klebsiella. Results Thrombin inhibition by dabigatran enhanced bacterial outgrowth and spreading, and accelerated mortality. Thrombin inhibition did not influence neutrophil recruitment to the lung or activation or neutrophil extracellular trap formation. Dabigatran reduced D-dimer formation and fibrin deposition in the lung. Fibrin depletion also enhanced bacterial outgrowth and spreading, and thrombin inhibition had no additional effect. Both thrombin and fibrin polymerization inhibited ex vivo Klebsiella outgrowth in human whole blood, which was neutrophil dependent, and the effect of thrombin required the presence of platelets and platelet protease activated receptor-1. In vivo thrombin inhibition reduced platelet-neutrophil complex formation and endothelial cell activation, but did not prevent sepsis-induced thrombocytopenia or organ damage. Conclusions These results suggest that thrombin plays an important role in protective immunity during pneumonia-derived sepsis by fibrin polymerization and enhancement of platelet-neutrophil interactions.

Measurable Amount of Active Thrombin Is Bound to Circulating D-Dimers. Is There Any Impact on Diagnosis and Pathophysiology of Thrombosis?

IntroductionThrombosis is a common pathology underlying ischemic heart disease, stroke, and venous thromboembolism. D-dimer is a global indicator of blood coagulation activation and fibrinolysis and, therefore, an indirect marker of thrombotic activity. D-dimers half-life is 48 hours. D-dimer determination is the standard procedure in the treatment of thrombosis. Serine protease thrombin plays pivotal roles in thrombosis and hemostasis, blood coagulation and platelet activation. Thrombin has a short half-life and naturally occurring inhibitors, such as antithrombin, rapidly bind thrombin, which makes the direct measurements of in vivo active thrombin difficult. Thrombin binds to fibrin where it is quite efficiently protected from inhibition by heparin-antithrombin but susceptible to inactivation by different antithrombin-independent inhibitors. There are two low affinity non-substrate thrombin binding sites, one in each half of the dimeric fibrinogen E region, and one high affinity thrombin non-substrate binding site on fibrinogen gamma' chains inside the D region (Meh DA, Siebenlist KR, Mosesson MW, J Biol Chem. 1996, 23121-5). We have shown that thrombin bound to fibrin promotes further fibrin growth in the presence of fibrinogen and absence of free thrombin in solution (Riedel T, Brynda E, Dyr JE, Houska, M, J. Biomed. Mater. Res. Part A 2009, 437-447). The aim of this project was to look for any thrombin activity on isolated D-dimers using several commercial D-dimer kits in groups of patients with elevated D-dimers.MethodsThree D-dimers kits were used (ELISA kits CEA506Hu, USCN and D-Dimer (D2D), BioAssay™; and immunoturbidimetric assay D-Dimer KAI-090, K-ASSAY). To detect thrombin activity on captured D-dimers one chromogenic (S-2238, Chromogenix) and two fluorogenic (SN-59, Haematologic Technologies, Inc.; (p-tosyl-Gly-Pro-Arg)2-R110, Thermo Fisher Scientific, Inc.) specific substrates and specific inhibitors (hirudine and PPACK) were used. Independently, bound proteins were removed from immobilized antibodies in 8 M urea and after treatment with trypsin analyzed by mass spectrometry (TripleTOF 5600, Sciex). 34 patients with high, moderate, and low levels of D-dimers and with different diagnosis were analyzed.ResultsOut of 18 patients with main diagnosis linked with thrombosis 61 % exhibited active thrombin on D-dimers. In these patients we found active thrombin bound to D-dimers captured by antibodies in all applied D-dimer kits. Using mass spectrometry thrombin bound to isolated D-dimers was detected. Specificity of thrombin activity related to D-dimers was determined by combination of several specific thrombin substrates and inhibitors. The activity of bound thrombin was remarkably stable over the period of more than 24 hours. It showed that precise measurement of even very low activity of thrombin was possible. In the group of 16 non-thrombotic patients with elevated D-dimers only 19 % exhibited thrombin activity. Interestingly, differences in values of thrombin activity were up to five orders of magnitude and differences in the activities related to the value of captured D-dimers were even greater.ConclusionThis, to our knowledge, is the first report showing the presence of active thrombin bound to circulating D-dimers. Although the group of patients we were so far able to evaluate is too small to being statistically tested, the results are highly encouraging. The amount of bound active thrombin on fibrin degradation products reflects the way of thrombus formation and its degradation (times, durations and rates of relevant reactions and especially the rate of thrombin generation). The thrombin concentration present at the time of fibrin gelation plays an important role in formation of fibrin clot structure, including its mechanical and fibrinolytic stability. It remains to be seen in further studies with much larger cohorts of patients if the presence of active thrombin has any impact on pathophysiology of thrombosis and if its determination may be of importance for the improvement of diagnosis of thrombotic events.Supported by the project of the Ministry of Health of the Czech Republic for conceptual development of the research organization 00023736, by Grant from the Academy of Sciences, Czech Republic (P205/12/G118), and by ERDF OPPK CZ.2.1.16/3.1.00/28007. DisclosuresNo relevant conflicts of interest to declare.

Physiologic variations in blood plasminogen levels affect outcomes after acute cerebral thromboembolism in mice: a pathophysiologic role for microvascular thrombosis

Essentials Physiologic variations in blood plasminogen (Pg) levels may affect ischemic stroke outcomes. We tested Pg effects in a model with translational relevance to human thromboembolic stroke. A dose-response exists between Pg levels and brain injury, fibrinolysis, barrier breakdown. Higher Pg levels reduce microvascular thrombosis and improve outcomes in ischemic stroke. Background and Objectives Plasminogen appears to affect brain inflammation, cell movement, fibrinolysis, neuronal excitotoxicity, and cell death. However, brain tissue and circulating blood plasminogen may have different roles, and there is wide individual variation in blood plasminogen levels. The aim of this study was to determine the integrated effect of blood plasminogen levels on ischemic brain injury. Methods We examined thromboembolic stroke in mice with varying, experimentally determined, blood plasminogen levels. Ischemic brain injury, blood-brain barrier breakdown, matrix metalloproteinase-9 expression and microvascular thrombosis were determined. Results Within the range of normal variation, plasminogen levels were strongly associated with ischemic brain injury; higher blood plasminogen levels had dose-related, protective effects. Higher plasminogen levels were associated with increased dissolution of the middle cerebral artery thrombus. Higher plasminogen levels decreased blood-brain barrier breakdown, matrix metalloproteinase-9 expression and microvascular thrombosis in the ischemic brain. In plasminogen-deficient mice, selective restoration of blood plasminogen levels reversed the harmful effects of plasminogen deficiency on ischemic brain injury. Specific inhibition of thrombin also reversed the effect of plasminogen deficiency on ischemic injury by decreasing microvascular thrombosis, blood-brain barrier breakdown, and matrix metalloproteinase-9 expression. Conclusions Variation in blood plasminogen levels, within the range seen in normal individuals, had marked effects on experimental ischemic brain injury. Higher plasminogen levels protected against ischemic brain injury, and decreased blood-brain barrier breakdown, matrix metalloproteinase-9 expression, and microvascular thrombosis. The protective effects of blood plasminogen appear to be mediated largely through a decrease in microvascular thrombosis in the ischemic territory.