Activated Protein C Resistance in Patients with Pre-Eclampsia in Lagos, Nigeria

Background: Preeclampsia is reported to complicate 2% - 8% of pregnancies globally and is an important cause of maternal and perinatal morbidity and mortality. The aetiology and pathogenesis are still poorly understood and substantial improvement has not been made in the prediction, prevention and treatment of the disease. Objective: To compare the frequency of activated protein C resistance (APC-R) in patients with pre-eclampsia to that of normotensive pregnant women and to determine the correlation between activated protein ratio (APC-ratio) and the severity of pre-eclampsia. Methodology: A cross-sectional study was carried out in 100 pre-eclamptic patients and 100 normotensive pregnant controls. The APC-ratio was determined using the modified activated partial thromboplastin time. Study participants with APC-ratio of less than 2.0 were defined as having APC-R. Data was analyzed using SPSS version 22.0. Results: Mean APC-ratio was significantly lower in pre-eclamptics (2.89 ± 1.70) compared to normotensive pregnant women (3.57 ± 1.06) (p = 0.0008) and the levels were also higher in mild (2.95 ± 1.15) compared to severe pre-eclamptics (2.62 ± 1.14). The frequency of APC-R was 26% among women with pre-eclampsia compared to 4% among normotensive controls (p = 0.000). Among 100 pre-eclamptic women 7 (21.2%) out of 33 with mild pre–eclampsia had APC-R, while 19 (28.4%) out of 67 with severe pre-eclampsia had APC-R. APC-ratio had a significant negative correlation with mean arterial blood pressure (r = −0.324;p = 0.000) and proteinuria (r = −0.379;p = 0.000) among study participants. Conclusion: The frequency of activated protein c resistance is significantly higher in pre-eclamptics compared to normotensive pregnant women and this is more pronounced in those with severe pre-eclampsia compared with those with mild disease. APC-R may therefore be used as a marker of severity in the disease.

Protective Effects of Activated Protein C on Neurovascular Unit in a Rat Model of Intrauterine Infection-Induced Neonatal White Matter Injury

Summary： Activated protein C （APC）, a natural anticoagulant, has been reported to exert direct vascu- loprotective, neural protective, anti-inflammatory, and proneurogenic activities in the central nervous system. This study was aimed to explore the neuroprotective effects and potential mechanisms of APC on the neurovascular unit of neonatal rats with intrauterine infection-induced white matter injury. In- traperitoneal injection of 300 ~tg/kg lipopolysaccharide （LPS） was administered consecutively to preg- nant Sprague-Dawley rats at embryonic days 19 and 20 to establish the rat model of intrauterine infec- tion-induced white matter injury. Control rats were injected with an equivalent amount of sterile saline on the same time. APC at the dosage of 0.2 mg/kg was intraperitoneally injected to neonatal rats imme- diately after birth. Brain tissues were collected at postnatal day 7 and stained with hematoxylin and eo- sin （H＆E）. Immunohistochemistry was used to evaluate myelin basic protein （MBP） expression in the periventricular white matter region. Blood-brain barrier （BBB） permeability and brain water content ~were measured using Evens Blue dye and wet/dry weight method. Double immunofluorescence staining and real-time quantitative PCR were performed to detect microglial activation and the expression of protease activated receptor 1 （PAR1）. Typical pathological changes of white matter injury were ob- served in rat brains exposed to LPS, and MBP expression in the periventricular region was significantly decreased. BBB was disrupted and the brain water content was increased. Microglia were largely acti- vated and the mRNA and protein levels of PAR1 were elevated. APC administration ameliorated the pathological lesions of the white matter and increased MBP expression. BBB permeability and brain water content were reduced. Microglia activation was inhibited and the PAR1 mRNA and protein ex- pression levels were both down-regulated. Our results suggested that APC exerted neuroprotective ef- fects on multiple components of the neurovascular unit in neonatal rats with intrauterine infec- tion-induced white matter injury, and the underlying mechanisms might involve decreased expression of PAR1.

Liver myofibroblasts activate protein C and respond to activated protein C

AIM:To study the protein C activation system in human liver myofibroblasts,and the effects of activated protein C(APC)on these cells.METHODS:Human liver myofibroblasts were obtained by outgrowth.Expression of protease activated receptor 1(PAR-1),endothelial protein C receptor(EPCR) and thrombomodulin(TM)was analyzed by flow cytometry.Extracellular signal-regulated kinase(ERK)1/2 activation was assessed by Western blotting using anti-phospho-ERK antibodies.Collagen synthesis was studied with real-time reverse transcription-polymerase chain reaction(RT-PCR).Activation of protein C was studied by incubating liver myofibroblasts with zymogen protein C in the presence of thrombin and detecting the generation of APC with a colorimetric assay using a peptide substrate. RESULTS:Primary cultures of human liver myofibroblasts expressed EPCR on their surface,together with PAR-1 and TM.This receptor system was functional since exposure of myofibroblasts to APC inducedERK1/2 phosphorylation in a dose-and time-dependent manner.Furthermore,APC significantly upregulated the expression of collagen mRNA,as shown by real-time RT-PCR.Collagen upregulation was controlled through the ERK pathway as it was inhibited when using the mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor PD98059.Finally,using a cell-based colorimetric assay,we showed that intact myofibroblasts converted protein C into APC in the presence of thrombin.CONCLUSION:These data suggest that APC is a new modulator of liver myofibroblast activity and contributes to the pathophysiology of chronic liver 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.

Effects of activated protein C on coagulation and fibrinolysis in rabbits with endotoxin induced acute lung injury

Background Sepsis induced acute lung injury （ALI） as a common syndrome in clinical practice has a high mortality. Recombinant human activated protein C （APC） can significantly reduce the mortality of patients with severe sepsis. Several studies have implicated that APC may be protective in ALI. Methods Twenty-one rabbits were operatively prepared and randomly divided into sham, control, or APC groups （n=7 in each group）. After a tracheotomy had been performed, ALI was produced in the control and APC groups by infusion of Escherichia coil endotoxin 100 μg/kg per hour intravenously for 1 hour. The sham group received only the vehicle, infusion of 20 ml of 0.9% saline. The rabbits were studied under anesthesia for 6 hours and were ventilated with 40% oxygen. Bovine APC （25 μg·kg^-1·h^-1） was intravenously administered. The infusion was initiated half an hour post-injury and lasted for 4 hours. The animals were resuscitated with Ringer＇s lactate solution. Results In comparison with nontreatment in the control group, the infusion of APC significantly reduced the increase of thrombomodulin level （TM; control group was （0.68±0.06） ng/ml, vs APC group of （0.62±0.07） ng/ml at 6 hours, P 〈0.05）, and significantly attenuated the fall in protein S （PS; control group was （2.32±0.03） μg/ml at 2 hours, （2.24±0.06） μg/ml at 4 hours and （2.21±0.09）μg/ml at 6 hours, vs APC group （2.46±0.04） μg/ml at 2 hours, （2.40±0.05） μg/ml at 4 hours and （2.39±0.07） μg/ml at 6 hours, P 〈0.01）. In addition, APC limited the increase in plasminogen activator inhibitor-1 （PAI-1） both in plasma （control group was （0.68±0.12） ng/ml at 1 hour, （0.84±0.06） ng/ml at 2 hours, （0.87±0.08） ng/ml at 4 hours and （0.91±0.05） ng/ml at 6 hours, vs APC group （0.42±0.16） ng/ml at 1 hour, （0.43±0.04） ng/ml at 2 hours, （0.45±0.09） ng/ml at 4 hours and （0.45±0.14） ng/ml at 6 hours, P 〈0.01） and in bronchoalveolar lavage fluid （at 6 hours： sham, （1.05±0.05） ng/ml; control, （1.13±0.06） ng/ml; APC, （1.06±0.06） ng/ml; P 〈0.05）. However, APC failed to prevent the decrease in PaO2/FiO2 ratio. APC-treated rabbits showed no significant difference in platelet count and antithrombin but exhibited less D-dimer production than did the controls. Moreover, APC limited the histopathological score of lung injury （2.6±0.8 in control, vs 1.4±0.6 in APC group, P〈0.01）. Conclusion Anti-coagulation and pro-fibrinolysis activity may be two of the possible mechanisms by which activated protein C attenuated endotoxin-induced ALI.

Developmental Lead Exposure Alters the Distribution of Protein Kinase C Activity in the Rat Hippocampus

Chronic low-level lead (Pb) exposure in children is known to cause a deficit in learning and memory. In vitro studies have demonstrated that Pb altered protein kinase C (PKC) activityt Especially, hippocampal PKC has been correlated with performance in several learning tasks. The effects of Pb exposure on hippocampal PKC were investigated during development at various postnatal ages: postnatal day (PN) 7, 14, 28, and 56. Two-tenth % Pb acetate was administered to pregnant and lactating dams and then administered to weanling rats in drinking water. PKC activity was measured in both membrane and cytosolic fractions from the hippocampi of the controls and Pb-exposed animals. Pb-induced increase in PKC activity in the cytosolic fraction was obsereved in the PN56 rats. In contrast, PKC activity was decreased by Pb at PN7 in the membrane fraction. Furthermore, a significant decrease in the ratio of membrane to cytosolic PKC activity which is representative of PKC distribution was observed in the PN28 and PN56 Pb-exposed rats relative to the same-age controls. This study indicates that chronic Pb exposure during development influences hippocampal PKC activity and distribution. These changes may be involved in the subclinical neurotoxicity of chronic Pb exposure in young children.

Antithrombin deficiency and decreased protein C activity in a young man with venous thromboembolism： a case report

Antithrombin and protein C are two crucial members in the anticoagulant system and play important roles in hemostasis. Mutations in SERPINC1 and PROC lead to deficiency or dysfunction of the two proteins, which could result in venous thromboembolism （VTE）. Here, we report a Chinese 22-year-old young man who developed recurrent and serious VTE in cerebral veins, visceral veins, and deep veins of the lower extremity. Laboratory tests and direct sequencing of PROC and SERPINC1 were conducted for the patient and his family members. Coagulation tests revealed that the patient presented type I antithrombin deficiency combined with decreased protein C activity resulting from a small insertion mutation c.848_849insGATGT in SERPINC1 and a short deletion variant c.572 574delAGA in PROC. This combination of the two mutations was absent in 400 healthy subjects each from southern and northern China. Then, we summarized all the mutations of the SERPINC1 and PROC gene reported in the Chinese Han population. This study demonstrates that the combination of antithrombin deficiency and decreased protein C activity can result in severe VTE and that the coexistence of different genetic factors may increase the risk of VTE.