The cardioprotective effects of marrubiin, a diterpenoid found in Leonotis leonurus extracts

Ethnopharmacological relevance

Leonotis leonurus L. (Lamiaceae) is used as a traditional medicine for a variety of ailments in South Africa. The diterpene marrubiin is the major product constituent in specimens of this plant occurring in South Africa.

Materials and methods

Marrubiin isolated from South African specimens of L. leonurus in addition to an organic extract of L. leonurus were tested in vivo, ex vivo and in vitro for their anticoagulant, antiplatelet and anti-inflammatory activities.

Results

Marrubiin and the organic extract suppressed coagulation, platelet aggregation and inflammatory markers. For the coagulation markers it was found that the organic extract and marrubiin significantly prolonged activated partial thromboplastin time (APTT). Fibrin and D-dimer formation were drastically decreased. These findings were observed in an ex vivo model and an obese rat model. Chemokines enhance leukocyte recruitment to inflammatory sites. TNF-α and RANTES secretion were significantly reduced by the extract and marrubiin when determined in the obese rat model relative to the controls. Calcium mobilization and TXB₂ synthesis were suppressed by the extract and marrubiin. An in vitro model was used to elucidate the antiplatelet mechanism and it was found that the extract and marrubiin inhibited platelet aggregation by inhibiting the binding of fibrinogen to glycoprotein (GP) IIb/IIIa receptor in a concentration dependent manner.

Conclusion

The findings reflect that marrubiin largely contributes to the extract's anticoagulant, antiplatelet and anti-inflammatory effects observed.     

Differential ability of tissue factor antibody clones on detection of tissue factor in blood cells and microparticles

Introduction

Tissue factor (TF), the primary initiator of coagulation in vivo, plays a major role in both thrombosis and hemostasis. The expression of TF in monocytes is well documented, but its presence in other blood cells has been disputed, possibly due to methodological variations among different studies.

Materials and methods

We studied TF expression on platelets, monocytes, lymphocytes and microparticles (MPs) by flow cytometry (FCM) with five commercially available mouse anti-human TF antibodies (HTF-1, TF9-10H10, CLB/TF-5, VIC7 and VD8). The ability of different TF antibodies to inhibit cell surface TF activity was explored by incubating LPS-stimulated monocytes and MPs derived from LPS-stimulated monocytes (MMPs) with TF antibodies followed by measuring TF activity.

Results

HTF-1 detected TF only on LPS-stimulated monocytes, whereas, TF9-10H10 and VD8 detected TF associated with MPs and MMPs in addition to LPS stimulated monocytes. Surprisingly, CLB/TF-5 and VIC7 detected TF on platelets, monocytes even under unstimulated conditions, in addition to MPs and MMPs. CLB/TF-5 also detected TF on unstimulated lymphocytes. Inhibitory studies showed that at a final concentration of 10 μg/mL, HTF-1, CLB/TF-5 and VD8 inhibited monocyte TF activity by 81-84% and MMP TF activity by 92-96%; whereas TF9-10H10 had no inhibitory effect on TF activity in monocytes and MMPs.

Conclusions

Our results suggest non-specific binding by the CLB/TF-5 and VIC7 antibodies in a FCM test system and explain at least some of the reports on TF presence in blood cells, particularly TF associated with platelets and MPs. TF9-10H10 and VD8 are more suitable to detect TF on MPs by FCM.     

Periodontopathogens induce expression of CD40L on human platelets via TLR2 and TLR4

Introduction

The outstanding importance of (soluble) CD40L to cardiovascular disease (CVD) is becoming increasingly apparent as CD40L is an important mediator of thrombotic and inflammatory processes. Platelets are the main source for CD40 ligand, linking platelet stimulatory events to inflammation and adverse adaptive immune responses. Periodontitis represents a chronic dental infection by distinct gram negative bacteria that is associated with an increased risk for CVD. However, the effects of periodontopathogens on CD40L expression by platelets have not been determined.

Material and Methods

Effects of periodontopathogens A. actinomycetemcomitans Y and P. gingivalis on the expression of CD40L were determined and the underlying receptors and pathways were investigated. 26 patients with periodontitis and 19 controls were included in the clinical part of this study.

Results

Periodontopathogens directly induce surface expression of CD40L in human platelets. This activation depends on plasma factors like CD14 and involves TLR2 and TLR4 but not FcγRII. Inhibition of PI3K and PLC completely abolishes bacteria-induced surface expression of CD40L. TLR2 and TLR4 agonists, for example, are also able to induce expression and release of CD40L in human platelets.In patients with periodontitis, plasma levels of soluble CD40L are elevated and positivity for P. gingivalis is associated with a statistical significant increase of soluble CD40L.

Conclusions

Our data indicate an involvement of periodontopathogens in increased plasma levels of soluble CD40L in periodontitis and therefore provide a novel link between periodontitis and increased risk for CVD.     

Platelet-rich plasma plus bioactive glass in the treatment of intra-bony defects: a study in dogs

Objective

This study was designed to evaluate, histomorphometrically, the association of platelet-rich plasma (PRP) and bioactive glass (BG) in the treatment of periodontal intrabony defects.

Material and Methods

Nine mongrel dogs were included in the study. Three-wall intrabony defects were surgically created at the mesial and distal aspect of first mandibular molar and exposed to plaque accumulation for 1 month. The defects were randomly assigned to the groups: control, BG, PRP, PRP+BG. Dogs were sacrificed 90 days after the surgeries. The histometric parameters evaluated were: length of sulcular and junctional epithelium, connective tissue adaptation, new cementum, new bone, defect extension and area of new bone filling the defect.

Results

A superior area of new bone was observed in PRP+BG and BG (13.80±2.32 mm² and 15.63±2.64 mm², respectively) when compared to the other groups (8.19±1.46 mm² and 8.81±1.47 mm² for control and PRP, respectively). No statistically significant differences were observed in the remaining parameters.

Conclusion

Within the limits of this study, it may be concluded that PRP failed to provide statistically significant improvements in the histometric parameters.     

Étude comparative de cinq techniques de préparation plaquettaire (platelet-rich plasma)

Aim of the study

Injections of platelet-rich plasma (PRP) constitute a new therapeutic for treating chronic tendinopathies. The injection being carried out in the tendon, the volume of PRP should thus be minimal (to decrease the intratendinous pressure and to minimize pain). This PRP should also have a raised platelet count. The quantity of released growth factors could be related to the system of preparation employed. We thus carried out a comparative study of five techniques of preparation of PRP described in the literature.

Materials and methods

Samples of venous blood were taken among five patients in order to compare five techniques of preparation of PRP: University Hospital of Liège technique, Curasan^® PRP Kit, Plateltex^®, GPS^®II and RegenLab^®.

Results

The various techniques make it possible to obtain more important platelet concentration than in blood, with variable volumes (0,3 to 6 ml). The number of platelets per microlitre appears higher with Plateltex^® and obtains smallest volume of PRP. The other techniques also give small volumes except for the GPS^®II. The number of collected platelets with this technique appears thus higher. The best collect efficiency is obtained with RegenLab^®.

Conclusion

The technique Plateltex^® makes it possible to collect the highest concentration of platelets in the smallest volume available.     

Engineered human soluble calcium-activated nucleotidase inhibits coagulation in vitro and thrombosis in vivo

Human soluble calcium-activated nucleotidase (human SCAN) is a homologue of the salivary anti-coagulant apyrases injected by insects into their hosts to allow blood feeding. However, the human enzyme, unlike its insect counterparts, does not efficiently hydrolyze the platelet agonist, ADP. By site-directed mutagenesis, two mutant human SCANs were constructed and expressed in bacteria. Following refolding from inclusion bodies and purification, these enzymes were assessed for anti-coagulant and anti-thrombotic efficacy. These engineered proteins include both active site mutations and a dimer interface mutation to increase the stability and ADPase activity of the modified human nucleotidase. The ADPase activity of these mutants increased more than ten fold. The E130Y/K201M/E216M SCAN mutant efficiently inhibited platelet aggregation in vitro. In addition, the E130Y/K201M/T206K/T207E/E216M mutant inhibited jugular vein thrombosis in the murine ferric chloride-induced model of thrombosis, as assessed by laser Doppler blood flow measurements. The bed bug insect homologue of human SCAN was also expressed and purified, and used in these in vivo experiments as a benchmark to assess the therapeutic potential of the engineered human enzymes. The most active modified human enzyme was able to completely inhibit the thrombosis induced by ferric chloride at roughly double the protein dose used for the bed bug enzyme. Thus, for the first time, we show that an engineered form of this human protein is efficacious in an in vivo model of thrombosis, demonstrating that suitably modified human SCAN enzymes have therapeutic potential as anti-coagulant and anti-thrombotic therapeutic agents. This suggests their utility in future treatment strategies for thrombotic cardiovascular diseases, including myocardial infarctions and ischemic strokes.     

Effects of bortezomib on platelet aggregation and ATP release in human platelets, in vitro

INTRODUCTION: Proteasome inhibitor bortezomib (PS-341) has been the first proteasome inhibitor that has entered clinical trials with its antiproliferative and proapoptotic effects in patients with multiple myeloma. Recent studies indicate that proteasome inhibitors can be useful in prevention of experimental arterial thrombosis in renovascular hypertensive rat models. The aim of the present study is to investigate the effect of bortezomib on in vitro platelet aggregation and adenosine triphosphate (ATP) release of human platelets. MATERIALS AND METHODS: For this purpose, platelet aggregation was induced in the platelet-rich plasma (PRP) using 3 microg ml(-1) collagen, 5 microM adenosine diphosphate (ADP), 10 microM epinephrine and 1 U ml(-1) thrombin and ATP release was induced by collagen. RESULTS AND CONCLUSIONS: Bortezomib showed an inhibitory effect on platelet aggregation induced by ADP in human PRP in a dose- and time-dependent manner, whereas it had no effect on collagen-, epinephrin and thrombin-induced aggregation. ATP-release reaction induced by collagen was inhibited dose- and time-dependently by bortezomib, even though collagen-induced platelet aggregation was apparently not affected in human PRP. These findings indicate that bortezomib may be an antiaggregating agent and its' effects may be related to adenine nucleotide receptor dependent regulatory proteins which are important for physiological and pathophysiological cellular processes. However, our in vitro studies suggest that this hypothesis is inadequate to explain the observations completely. This phenomenon and its clinical implication justify further clinical investigations.     

Alteration in protein kinase B (AKT) activity in platelets from patients with systemic sclerosis

We have previously reported that there is an increase in phosphotidylinositide-3 kinase activity secondary to increase in nitrotyrosylation in platelets from patients with SSc. Protein kinase B (Akt) is recruited to the plasma membrane by PI 3-K metabolites. Both enzymes play critical roles in signal transduction and activation of platelets. In the present investigation, we have studied the effect of postranslational modification of the activity of Akt. We have examined eight patients and eight controls and obtained results showing that enzymatic activity of Akt is increased in lysates of platelets from patients with SSc compared to normal volunteer controls. We have obtained results showing that there is no correlation of nitrotyrosylation of Akt on its enzymatic activity although Western blots show the enzyme has increased nitrotyrosylation. These results suggest that post-translational modification by nitrotyrosylation does not control Akt in SSc platelets. We concluded that the enhanced activity of PI 3-K and Akt in platelets from patients with SSc is mediated by different mechanisms.     

Prediction of the therapeutic index of marketed anti-coagulants and anti-platelet agents by guinea pig models of thrombosis and hemostasis

Introduction

Animal models of thrombosis and hemostasis are critical for target validation in pharmaceutical research. Guinea pig haemostatic mechanisms, such as the platelet thrombin receptor repertoire, resemble those of humans. Measuring the performance characteristics of marketed antithrombotic drugs in guinea pig models is a key to predicting therapeutic indices of new agents. The goal of the current study was to benchmark representative marketed drugs in thrombosis and hemostasis models in guinea pigs.

Methods

Effects of the cyclooxygenase inhibitor, aspirin, the P2Y₁₂ antagonist, clopidogrel, the glycoprotein IIb/IIIa inhibitor, tirofiban, and the direct thrombin inhibitors, argatroban and hirudin, were evaluated in this study. Antithrombotic agents were tested in FeCl₃-induced carotid artery thrombosis and arterio-venous shunt thrombosis models. Haemostatic effects of drugs were evaluated in cuticle and renal bleeding models. Ex vivo measurements of platelet function and coagulation inhibition were performed to benchmark preclinical doses of each agent to those used clinically.

Results

The overall rank-order of potency in thrombosis models based on per cent of vessels occluded, average carotid blood flow, and thrombus weight was aspirin = argatroban = tirofiban < hirudin = clopidogrel. In bleeding models, the rank order was: aspirin < clopidogrel = argatroban = tirofiban < hirudin.

Conclusion

This characterization of representative drugs from two important classes of anti-coagulant and anti-platelet agents in efficacy and bleeding models in guinea pigs provides a reference point for evaluation of new antithrombotic agents.     

b型流感嗜血杆菌结合疫苗接种后婴幼儿安全性和免疫原性研究

目的 评价b型流感嗜血杆菌结合疫苗(Hib-TT)安全性和免疫原性.方法 分别采用Hib-TT试验疫苗和对照疫苗3针免疫接种3～5月龄婴幼儿,观察疫苗安全性,并采用定量ELISA法分别测定免疫前、免疫后和加强免疫后血清特异性IgG抗体浓度.结果 实验疫苗和对照疫苗两组间不良反应总发生率(实验疫苗组为23.85％,对照疫苗组为31.40％)差异无统计学意义(x2=0.5,P＞0.05),发热性总不良反应率分别为22.3％和31.3％,中、强发热反应率分别为3.67％和4.48％,差异无统计学意义;实验疫苗受试者局部红、肿、硬结等不良反应率为1.22％.实验疫苗3剂免疫后受试者血清抗Hib PRP IgG抗体平均几何浓度(GMC)为6.6786 μg/ml,对照疫苗组血清抗体GMC为7.5346 μg/ml,两组间抗体GMC差异无统计学意义(x2=0.147,P=0.702);加强免疫1剂后,实验疫苗组受试者血清抗体GMC从加强免疫前的2.6396 μg/ml上升为6.2044μg/ml.结论 实验疫苗接种3～5月龄婴幼儿具有良好的安全性.用间隔1个月、3剂次接种的基础免疫程序能诱导该年龄组受试者产生长期保护水平的血清特异性抗体,加强免疫1剂后能诱导机体产生免疫记忆反应.