Aggregation In Platelet-rich Plasma Research Articles

Platelet Carbonic Anhydrase II, a Forgotten Enzyme, May Be Responsible for Aspirin Resistance.

Background Thromboembolic events constitute a major health problem, despite the steadily expanding arsenal of antiplatelet drugs. Hence, there is still a need to optimize the antiplatelet therapy. Objectives The aim of our study was to verify a hypothesis that there are no differences in platelet proteome between two groups of healthy people representing different acetylsalicylic acid (aspirin) responses as assessed by the liquid chromatography/mass spectrometry (LC/MS) technique. Patients/Methods A total of 61 healthy volunteers were recruited for the study. Physical examination and blood collection were followed by platelet-rich plasma aggregation assays and platelet separation for proteomic LC/MS analysis. Arachidonic acid- (AA-) induced aggregation (in the presence of aspirin) allowed to divide study participants into two groups aspirin-resistant (AR) and aspirin-sensitive (AS) ones. Subsequently, platelet proteome was compared in groups using the LC/MS analysis. Results The LC/MS analysis of platelet proteome between groups revealed that out of all identified proteins, the only discriminatory protein, affecting aspirin responsiveness, is platelet carbonic anhydrase II (CA II). Conclusions CA II is a platelet function modulator and should be taken into consideration as a cardiovascular event risk factor or therapeutic target.

Anti-thrombotic and anti-tumor effect of water extract of caulis of <i>Sargentodoxa cuneata</i> (Oliv) Rehd et Wils (Lardizabalaceae) in animal models

Purpose: To investigate the anti-thrombosis and anti-tumor effect of the water extract of the caulis of Sargentodoxa cuneata (Oliv.) Rehd. et Wils. (WCSW) in rat and mouse models.Methods: WCSW extract was prepared and the main constituents were determined by high pressure liquid chromatography (HPLC). The acute toxicity of the extract was determined in mice. Platelet aggregation in rat platelet-rich plasma (PRP) was examined to evaluate the effect of the extract on platelet function. Thereafter, the cytotoxic activity of WCSW on HL60, A549, S180 and H22 cells was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In vivo antitumor effect of WCSW was further evaluated on H22 cells transplanted in mice, while the expression of caspase-3, caspase-9, Bcl-2 and Bax proteins were assayed by Western blot analysis.Results: Protocatechuic acid, rhodiola glucoside and chlorogenic acid were identified as the main constituents of WCSW. Platelet aggregation was significantly inhibited by treatment with the extract at concentrations of 1, 5 and 10 mg/mL. WCSW also showed significant inhibitory effect on HL60, A549, S180 and H22 cells in vitro with half maximal inhibitory concentration (IC50 value of 321.9, 285.0, 130.3 and 76.1 μg/mL, respectively. Furthermore, WCSW exhibited obvious anti-tumor effect on H22 transplanted tumor in vivo. After treatment with WCSW, caspase-3, caspase-9 and Bax were significantly (p < 0.05) up-regulated, whereas Bcl-2 was significantly (p < 0.05) down-regulated in the tumor tissues.Conclusion: WCSW possesses significant antithrombosis and anti-tumor effect, and therefore, has the potentials to be developed into effective drugs for clinical treatment of cancer and thrombosis diseases.Keywords: Sargentodoxa cuneata, Anti-thrombosis, Anti-tumor, Platelet aggregation, Apoptosis, Caspase, Protocatechuic acid, Rhodiola glucoside, Chlorogenic acid

The Effect of Hyperparathyroid State on Platelet Functions and Bone Loss.

Objective:Coagulation and fibrinolysis defects were reported in primary hyperparathyroid patients. However, there are not enough data regarding platelet functions in this group of patients. Our aim was to evaluate the platelet functions in primary and secondary hyperparathyroid patients and to compare them with healthy subjects.Materials and Methods:In our study 25 subjects with primary hyperparathyroidism (PHPT), 25 subjects with secondary hyperparathyroidism (SHPT), and 25 healthy controls were included. Platelet functions of the subjects were evaluated by using platelet-rich plasma and platelet aggregation tests induced with epinephrine, adenosine diphosphate (ADP), collagen, and ristocetin. Serum P selectin levels, which indicate platelet activation level, were measured in all subjects. Bone mineral densitometry was performed for all patients.Results:There was no significant difference between the groups with PHPT and SHPT and the control group regarding the platelet aggregation tests and serum P selectin levels. There was also no significant correlation between parathormone levels and aggregation parameters (ristocetin, epinephrine, collagen, and ADP: respectively p=0.446, 0.537, 0.346, and 0.302) and between P selectin (p=0.516) levels. When we separated the patients according to serum calcium levels, there was also no significant difference between aggregation parameters and serum P selectin levels between the patients with hypercalcemia and the patients with normocalcemia. We could not find any significant correlation between aggregation parameters, P selectin levels, and serum calcium levels in this group of patients. Bone loss was greater in patients with PHPT.Conclusion:There is no significant effect of PHPT or SHPT and serum calcium levels on platelet functions when evaluated by aggregation tests.

Synthesis, Anticlotting and Antiplatelet Effects of 1,2,3-Triazoles Derivatives.

The anticoagulant and antiplatelet aggregation potential of a series of six synthetic 1,2,3-triazole derivatives were investigated through in vitro models. Coagulation tests included the prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time (TT) assays, and were performed on a multichannel coagulometer, using human plasma. The platelet aggregation assays were carried out using human platelet-rich-plasma (PRP). Aggregation was initiated by adding ADP or collagen and monitored turbidimetrically on a Whole Blood Aggregometer. Toxicity of derivatives was evaluated on platelets and red blood cells, by measuring the release of lactate dehydrogenase and hemoglobin, respectively. Moreover, theoretical toxicity of derivatives was calculated using the software Osiris® Property Explorer. All the six derivatives tested inhibited, but with different potencies, the plasma coagulation assessed by the PT and TT assays, and also inhibited platelet aggregation of PRP induced by collagen or ADP. The derivatives did not interfere in the aPTT assay and did not affect the viability of platelets or red blood cells. Theoretical studies also revealed that all derivatives will likely to have low toxicity, great pharmacological and oral bioavailability profiles, and a Druglikeness and Drug score similar to some commercial anticoagulant and antiplatelet drugs. 1,2,3-triazoles are potential candidates for molecular modeling of new antithrombotic drugs.

RGDfK-functionalized gold nanorods bind only to activated platelets.

Integrin-targeting peptide RGDfK-labeled gold nanorods (GNR) seek to improve hyperthermia targeted to solid tumors by exploiting the known up-regulation of integrin αvβ3 cell membrane proteins on solid tumor vasculature surfaces. Tumor binding specificity might be expected since surrounding tissues and endothelial cells have limited numbers of these receptors. However, RGD peptide binding to many proteins is promiscuous, with known affinity to several families of cell integrin receptors, and also possible binding to platelets after intravenous infusion via a different integrin receptor, αIIbβ3, on platelets. Binding of RGDfK-targeted GNR could considerably impact platelet function, ultimately leading to increased risk of bleeding or thrombosis depending on the degree of interaction. We sought to determine if RGDfK-labeled GNR could interact with platelets and alter platelet function. Targeted and untargeted nanorods exhibited little interaction with resting platelets in platelet rich plasma (PRP) preparations. However, upon platelet activation, peptide-targeted nanorods bound actively to platelets. Addition of RGDfK-GNR to unactivated platelets had little effect on markers of platelet activation, indicating that RGDfK-nanorods were incapable of inducing platelet activation. We next tested whether activated platelet function was altered in the presence of peptide-targeted nanorods. Platelet aggregation in whole blood and PRP in the presence of targeted nanorods had no significant effect on platelet aggregation. These data suggest that RGDfK-GNR alone have little impact on platelet function in plasma. However, nonspecific nanorod binding may occur in vascular beds where activated platelets are normally cleared, such as the spleen and liver, producing a possible toxicity risk for these nanomaterials. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 209-217, 2017.

The effect of centrifugation speed and time on pre-analytical platelet activation

The results of laboratory analyses are affected by pre-analytical variables, and in particular can platelets be activated by shear handling stress and secrete granular substances. We therefore evaluated the effect of centrifugation speed and time on pre-analytical platelet activation. Citrate- and EDTA-anticoagulated blood from healthy volunteers were centrifuged at 80-10,000 g for 5-15 min to prepare plasma and platelet-rich plasma. Pre-analytical platelet activation was assessed by flow cytometric measurement of platelet P-selectin (CD62p) expression. Blood cell counts, mean platelet volume (MPV), immature platelet fraction (IPF), and platelet distribution width (PDW) were measured. Platelet aggregation in platelet-rich plasma induced by arachidonic acid (AA), ADP or thrombin receptor activator peptide-6 (TRAP) was tested by 96-well aggregometry. The median percentage of platelets expressing P-selectin in citrate- and EDTA-plasma centrifuged at 2000 g for 10 min were 43% [interquartile range (IQR), 38%-53%] and 56% (IQR, 31%-78%), respectively (p=0.82). Platelet-rich plasma prepared at 100-250 g for 10 min had significantly lower platelet P-selectin expression (11%-15%), p<0.001. Platelet count in plasma samples decreased with increasing speed but platelets were only completely removed if plasma was re-centrifuged. In platelet-rich plasma, increasing centrifugation speed significantly increased platelet yield but decreased contamination from other blood cells, platelet composition was altered as platelet parameters (MPV, IPF, and PDW) was lowered. Platelet aggregation was not affected by the centrifugation speed platelet-rich plasma was prepared. Proportional to centrifugation speed, platelets in plasma and platelet-rich plasma were activated with centrifugation speed, cell content and composition changed while platelet aggregation was unaltered.

Type 2B von Willebrand Disease: A Matter of Plasma Plus Platelet Abnormality.

Type 2B von Willebrand disease (VWD2B) is a rare, autosomal-dominant inherited bleeding disorder, characterized by an enhanced ristocetin-induced platelet aggregation in platelet-rich plasma and often with variable degree of thrombocytopenia and loss of high-molecular-weight multimers von Willebrand factor (VWF). All these phenomena are caused by a mutant VWF, normally synthesized and assembled by endothelial cells, but with heightened affinity binding to the platelet receptor glycoprotein Ib-α (GpIb-α). When this abnormal VWF is released into the circulation and under specific clinical circumstances, in vivo platelet clumping is observed. Mutations, invariably clustered in exon 28 of the VWF gene encoding for the VWF A1 domain involved in VWF binding to GpIb-α, are responsible for VWD2B phenotype. Clinical and laboratory phenotype appears strongly related to the type of VWF-causative mutations. However, recent evidences suggest that a true platelet defect is also present in this type, with several morphological and functional abnormalities being detected in a subset of VWD2B patients.

Escherichia coli induces platelet aggregation in an FcγRIIa‐dependent manner

The discovery of pathogen-recognition receptors such as Toll-like receptors on platelets has led to the emergence of the concept of platelets as important components of the host response to infection. Escherichia coli (E. coli)-mediated sepsis is a serious illness characterized by the occurrence of thrombocytopenia. Whereas there has been a wealth of research on platelet activation by Gram-positive bacteria, little is known about the mechanisms associated with Gram-negative bacteria-induced platelet activation with Gram-negative bacteria. To determine the mechanisms by which Gram-negative E. coli induces platelet aggregation. Induction of platelet aggregation with E. coli strain O157:H7 was tested in platelet-rich plasma (PRP), washed platelets, and serum depleted of complement factors. Platelet inhibitors (against αII b β3 , glycoprotein Ibα and FcγRIIa) were used. Platelet thromboxane synthesis was analyzed after E. coli stimulation. Cell binding assays were used to assess the ability of E. coli to support platelet adhesion. Trypsinization was used to determine the role of E. coli surface proteins. E. coli-induced aggregation in PRP was donor-dependent. E. coli O157:H7 induced aggregation with a lag time of 6.9 ± 1.3 min in an αII b β3 -dependent and FcγRIIa-dependent manner. Furthermore, this interaction was enhanced by the presence of complement, and was dependent on thromboxane synthesis. These results show E. coli to be a potent inducer of platelet aggregation.

Inhibition of complement component C5 prevents clotting in an ex vivo model of xenogeneic activation of coagulation.

Xenogeneic thrombotic microangiopathy (TMA) and acute vascular rejection (AVR) prevent long-term survival of porcine xenografts after transplantation into non-human primates. Preformed xenoreactive natural antibodies (XNA) cause endothelial damage and activate the complement system. Mechanisms of xenogeneic coagulation and platelet activation are only poorly characterized. A microfluidic flow chamber was used to study platelet activation and thrombus formation of human platelet-rich plasma (PRP) upon perfusion over wild-type (WT) or α-1,3- galactosyltransferase knockout (GTKO) and human CD46 (hCD46) transgenic porcine aortic endothelial cells (PAEC). Activation of plasma coagulation (thrombin-anti-thrombin complex; TAT) and complement (C3a, C5a) was studied in human platelet-free plasma (PFP) after co-incubation with PAEC. The activation of PAEC (E-Selectin, tissue factor, ICAM-1, ICAM-2, VCAM-1) was studied after incubation with human serum. Eculizumab (200 μg/ml) was used to inhibit terminal complement activation in all experiments. WT-PAEC perfused with human PRP showed thrombus formation at different shear rates (3 dyn/cm(2) : 23 ± 10%; 10 dyn/cm(2) : 17 ± 10% of flow chamber viewing field). GTKO/hCD46 PAEC exhibited reduced, but not fully prevented thrombus formation (3 dyn/cm(2) : 12 ± 12%). Porcine PRP caused little or no thrombus formation (3.0 ± 4% and 0.5 ± 0.9%, respectively). Flow cytometry of human platelets after perfusion over WT-PAEC revealed an increase in platelet CD62P expression (29.5 ± 3%), compared to non-perfused PRP (7 ± 2%) or PRP running through empty flow chambers (12.7 ± 0.3%). Incubation of human PFP with PAEC resulted in an increase of TAT that correlated with C5a activation. Specific inhibition of complement by eculizumab prevented thrombus formation (WT-PAEC: 1.6 ± 2% at 3 dyn/cm(2) and 0.24 ± 0.33% at 10 dyn/cm(2) , GTKO/hCD46 PAEC: 0.2 ± 0.3% at 3 dyn/cm(2) ) as well as activation of coagulation and platelets. Induction of endothelial E-Selectin and VCAM-1 in WT-PAEC upon incubation with human serum was significantly reduced by eculizumab. Eculizumab did not reduce thrombin generation capacity of human PRP or normal platelet aggregation. Thrombus formation in this ex vivo model of xenogeneic TMA was closely linked with complement activation. Specific inhibition of complement C5 by eculizumab prevented endothelial cell activation, but also coagulation and platelet activation without compromising thrombin generation capacity of human blood or normal platelet function.

Silica Nanoparticles Effects on Blood Coagulation Proteins and Platelets.

Interaction of nanoparticles with the blood coagulation is important prior to their using as the drug carriers or therapeutic agents. The aim of present work was studying of the primary effects of silica nanoparticles (SiNPs) on haemostasis in vitro. We studied the effect of SiNPs on blood coagulation directly estimating the activation of prothrombin and factor X and to verify any possible effect of SiNPs on human platelets. It was shown that SiNPs shortened coagulation time in APTT and PT tests and increased the activation of factor X induced by RVV possibly due to the sorption of intrinsic pathway factors on their surface. SiNPs inhibited the aggregation of platelet rich plasma induced by ADP but in the same time partially activated platelets as it was shown using flow cytometry. The possibility of SiNPs usage in nanomedicine is strongly dependant on their final concentration in bloodstream and the size of the particles that are used. However SiNPs are extremely promising as the haemostatic agents for preventing the blood loss after damage.

The anti-adhesive and anti-aggregatory effects of phenolics from Trifolium species in vitro.

The present in vitro study includes a comparative evaluation of anti-platelet (anti-thrombotic) properties of plant phenolics, isolated from nine different clover (Trifolium) species. The analysis covered phenolic fractions isolated from T. alexandrinum L., T. fragiferum L., T. hybridum L., T. incarnatum L., T. pallidum Waldst et Kit., T. resupinatum L. var. majus Boiss, T. resupinatum L. var. resupinatum, T. scabrum L., and T. pratense L. (red clover). The inhibitory effects of plant preparations (1–50 µg/ml) on hemostatic functions of blood platelets were assessed by measurements of thrombin- or ADP-induced platelet adhesion to fibrinogen, platelet aggregation in platelet-rich plasma (activated with ADP or collagen), and by the determination of PF-4 secretion from platelet α-granules. The influence of T. phenolics on arachidonic cascade in blood platelets was also determined. T. resupinatum var. majus, T. resupinatum var. resupinatum, and T. scabrum had the strongest anti-platelet effects. These preparations displayed the most evident anti-adhesive and anti-aggregatory effects in response to all of the used agonists: thrombin (0.2 U/ml), ADP (10 µM), and collagen (2 µg/ml), and their inhibitory properties were also confirmed by an analysis of PF-4 secretion. T. scabrum and some of other examined clover species possess significantly higher concentrations of both isoflavones and other bioactive phenolics, when compared to red clover. The obtained results suggest that these clovers contain substances with potent anti-platelet properties.

Platelet Activation and Aggregation in Patients with Advanced Adenocarcinoma Undergoing Chemotherapy: Correlation with a Validated Venous Thromboembolism Risk Score

Background: Cancer is known to increase risk of venous thromboembolism (VTE), which is associated with considerable mortality and morbidity. Chemotherapy is an independent risk factor for thrombosis in cancer patients. We examined platelet activation and reactivity based on the Khorana Score, a validated scoring model for VTE risk in patients receiving chemotherapy.Materials and Methods: Patients (n=25) with advanced stage adenocarcinomas (TNM stage III or greater), were enrolled in this study. Patients on antiplatelet therapy or on anticoagulation were excluded. Approximately 10 ml of Citrated blood samples were collected from central access catheters after discarding initial 5 ml to minimize iatrogenic platelet activation. 1μMol, 2μMol, 5μMol of ADP-induced, 2μMol Arachidonic Acid (AA)-induced and 4μMol Collagen-induced platelet aggregation in Platelet Rich plasma (PRP) was assessed with a light transmission aggregometry assay. Maximal aggregation and aggregation velocity were recorded. Concurrent flowcytometry analysis was done to assess the expressions of CD41 (Glycoprotein GpIIb), CD62p (platelet surface p-Selectin), and PAC-1(Activated GP IIb/IIIa).Thromboembolic risk scores (Khorana Scores, KS) were calculated based on a validated scoring system (Khorana AA, et al. Blood. 2008;111:4902-7) as follows: Table 1Very high risk cancers (pancreatic or gastric)+2 pointsHigh risk cancers (lung, ovarian, or bladder cancer)+1 pointPlatelet count ≥350 x 109/L+1 pointHemoglobin < 10 gm/dL or use of erythropoietin+1 pointLeukocytosis >11 x 109/L+1 pointBMI > 35+1 pointResults: Mean values for maximum platelet aggregation were calculated by groups and compared between patient with KS < 3 and KS > = 3 by two sample t-tests. Differences were statistically significant for all concentrations of ADP and collagen, with a positive trend for Arachidonic acid (Table). A significant linear relationship between maximal platelet aggregation and higher KS was observed. However there were no significant differences observed in the expression of platelet surface p-selectin, CD41 or PAC-1 when comparing patients at Khorana scores 0, 3 or more than 3 (not shown). Table 2Agonist ConcentrationKhorana Score <3Khorana Score >= 3P valueADP 1μMol20.8%28.14%0.02ADP 2μMol38.6%67%0.002ADP 5μMol66.5%79.85%0.006Arachidonic acid 2μMol61.6%73.85%0.12Collagen 4μMol78.6%90.14%0.01Discussion: The mechanism of cancer-related thromboembolism is not well understood. Chemotherapy is an added risk factor for the development of VTEs. Several new markers such as soluble p-selectin and mean platelet volume have been investigated as adjunct factors to improve predictive ability. Although our study did not find a correlation of some common platelet surface markers for activation with thrombotic risk scores; we find a strong positive correlation of future thrombotic risk derived via Khorana scores with heightened platelet reactivity. Platelet reactivity may mediate a final common pathway for venous thromboembolism in cancer patients and should be validated further as an adjunctive marker in a large scale study. DisclosuresNo relevant conflicts of interest to declare.

NOSH-aspirin (NBS-1120), a novel nitric oxide- and hydrogen sulfide-releasing hybrid has enhanced chemo-preventive properties compared to aspirin, is gastrointestinal safe with all the classic therapeutic indications

Aspirin is chemopreventive; however, side effects preclude its long-term use. NOSH-aspirin (NBS-1120), a novel hybrid that releases nitric oxide and hydrogen sulfide, was designed to be a safer alternative. Here we compare the gastrointestinal safety, anti-inflammatory, analgesic, anti-pyretic, anti-platelet, and chemopreventive properties of aspirin and NBS-1120 administered orally to rats at equimolar doses. Gastrointestinal safety: 6h post-administration, the number and size of hemorrhagic lesions in stomachs were counted; tissue samples were frozen for PGE2, SOD, and MDA determination. Anti-inflammatory: 1h after drug administration, the volume of carrageenan-induced rat paw edemas was measured for 5h. Anti-pyretic: fever was induced by LPS (ip) an hour before administration of the test drugs, core body temperature was measured hourly for 5h. Analgesic: time-dependent analgesic effects were evaluated by carrageenan-induced hyperalgesia. Antiplatelet: anti-aggregatory effects were studied on collagen-induced platelet aggregation of human platelet-rich plasma. Chemoprevention: nude mice were gavaged daily for 25 days with vehicle, aspirin or NBS-1120. After one week, each mouse was inoculated subcutaneously in the right flank with HT-29 human colon cancer cells. Both agents reduced PGE2 levels in stomach tissue; however, NBS-1120 did not cause any stomach ulcers, whereas aspirin caused significant bleeding. Lipid peroxidation induced by aspirin was higher than that exerted by NBS-1120. SOD activity was significantly inhibited by aspirin but increased by NBS-1120. Both agents showed similar anti-inflammatory, analgesic, anti-pyretic, and anti-platelet activities. Aspirin increased plasma TNFα more than NBS-1120-treated animals. NBS-1120 was better than aspirin as a chemopreventive agent; it dose-dependently inhibited tumor growth and tumor mass.

NOSH-sulindac (AVT-18A) is a novel nitric oxide- and hydrogen sulfide-releasing hybrid that is gastrointestinal safe and has potent anti-inflammatory, analgesic, antipyretic, anti-platelet, and anti-cancer properties

Sulindac is chemopreventive and has utility in patients with familial adenomatous polyposis; however, side effects preclude its long-term use. NOSH-sulindac (AVT-18A) releases nitric oxide and hydrogen sulfide, was designed to be a safer alternative. Here we compare the gastrointestinal safety, anti-inflammatory, analgesic, anti-pyretic, anti-platelet, and anti-cancer properties of sulindac and NOSH-sulindac administered orally to rats at equimolar doses. Gastrointestinal safety: 6h post-administration, number/size of hemorrhagic lesions in stomachs were counted. Tissue samples were frozen for PGE2, SOD, and MDA determination. Anti-inflammatory: 1h after drug administration, the volume of carrageenan-induced rat paw edemas was measured for 5h. Anti-pyretic: fever was induced by LPS (ip) an hour before administration of the test drugs, core body temperature was measured hourly for 5h. Analgesic: time-dependent analgesic effects were evaluated by carrageenan-induced hyperalgesia. Antiplatelet: anti-aggregatory effects were studied on collagen-induced platelet aggregation of human platelet-rich plasma. Anti-cancer: We examined the effects of NOSH-sulindac on the growth properties of 12 human cancer cell lines of six different tissue origins. Both agents reduced PGE2 levels in stomach tissue; however, NOSH-sulindac did not cause any stomach ulcers, whereas sulindac caused significant bleeding. Lipid peroxidation induced by sulindac was higher than that from NOSH-sulindac. SOD activity was significantly lowered by sulindac but increased by NOSH-sulindac. Both agents showed similar anti-inflammatory, analgesic, anti-pyretic, and anti-platelet activities. Sulindac increased plasma TNFα whereas this rise was lower in the NOSH-sulindac-treated animals. NOSH-sulindac inhibited the growth of all cancer cell lines studied, with potencies of 1000- to 9000-fold greater than that of sulindac. NOSH-sulindac inhibited cell proliferation, induced apoptosis, and caused G2/M cell cycle block. These results demonstrate that NOSH-sulindac is gastrointestinal safe, and maintains the anti-inflammatory, analgesic, antipyretic, and antiplatelet properties of its parent compound sulinsac, with anti-growth activity against a wide variety of human cancer cells.

Ginkgo biloba Extract Inhibits Platelet Activation via Inhibition of Akt

Ginkgo biloba extract (GBE) contains flavone glycosides and terpenoids. It can modify vasomotor function, reduce the adhesion of blood cells to the endothelium, and inhibit the activation of platelets, and therefore plays an important role in the treatment of a variety of cardiovascular and neurological disorders. However, the molecular mechanism underlying its significant anticoagulation activity is largely unknown. In this study, we found that GBE adenosine diphosphate, collagen, and U4 (U46619) induced platelet aggregation in both platelet-rich plasma and washed platelets. GBE could reduce the adhesion of platelets to a collagen-coated surface and platelet spreading on fibrinogen. The treatment with GBE significantly inhibited the phosphorylation of Akt, suggesting that its inhibition of platelet activation might be mediated by attenuation of the PI3K/Akt pathway.

Substitution of the Echistatin Amino Acid Motif RGDD with KGDW Enhances Inhibition of Platelet Aggregation and Thrombogenesis

Disintegrins are a family of small proteins found in snake venom. These proteins are of great biomedical importance due to their binding affinities with different kinds of integrins, which results in the inhibition of platelet aggregation, the adhesion of cancer cells, and the induction of signal transduction pathways. Disintegrins are multi-functional due to their lower integrin selectivity. To increase their binding specificity with platelets, we modified the gene sequence encoding one kind of disintegrins, echistatin (Ech). DNA recombination technology was used to change the Ech amino acid sequence RGDD to KGDW, which are located at the 24th–27th location site of Ech, thereby creating a mutant protein echistatin (E-KW). The E-KW was expressed, isolated and purified using molecular biological methods. ADP-induced platelet aggregation in human platelet-rich plasma was inhibited by E-KW at 45 nM and by Ech at an IC50 of 140 nM (P < 0.05). E-KW was also more effective than wild-type Ech in inhibiting thrombus formation (12.8 ± 3.2 vs. 27.9 ± 4.1, P < 0.05). Our studies revealed that, compared to wild-type Ech, E-KW had stronger binding specificity to the glucoprotein receptors on platelet membranes and was more effective in inhibiting platelet aggregation and thrombogenesis.

In vitro pharmacological characterization of vorapaxar, a novel platelet thrombin receptor antagonist

Vorapaxar is a novel protease-activated receptor-1 (PAR1) antagonist recently approved for the reduction of thrombotic cardiovascular events in patients with a history of myocardial infarction or with peripheral arterial disease. The present study provides a comprehensive in vitro pharmacological characterization of vorapaxar interaction with the PAR1 receptor on human platelets. Similar studies were performed with a metabolite of vorapaxar (M20). Vorapaxar and M20 were competitive PAR1 antagonists that demonstrated concentration-dependent, saturable, specific, and slowly reversible binding to the receptor present on intact human platelets. The affinities of vorapaxar and M20 for the PAR1 receptor were in the low nanomolar range, as determined by saturation-, kinetic- and competitive binding studies. The calculated Kd and Ki values for vorapaxar increased in the presence of plasma, indicating a decrease in the free fraction available for binding to the PAR1 receptor on human platelets. Vorapaxar was also evaluated in functional assays using thrombin or a PAR1 agonist peptide (SFLLRN). Vorapaxar and M20 completely blocked thrombin-stimulated PAR1/β-arrestin association in recombinant cells and abolished thrombin-stimulated calcium influx in washed human platelets and vascular smooth muscle cells. Moreover, vorapaxar and M20 inhibited PAR1 agonist peptide-mediated platelet aggregation in human platelet rich plasma with a steep concentration response relationship. Vorapaxar exhibited high selectivity for inhibition of PAR1 over other platelet GPCRs. In conclusion, vorapaxar is a potent PAR1 antagonist exhibiting saturable, reversible, selective binding with slow off-rate kinetics and effectively inhibits thrombin’s PAR1-mediated actions on human platelets.

A primary role for kinin B1 receptor in inflammation, organ damage, and lethal thrombosis in a rat model of septic shock in diabetes.

Diabetes mellitus and septic shock increase the incidence of mortality by thrombosis. Although kinin B1 receptor (B1R) is involved in both pathologies, its role in platelet function and thrombosis remains unknown. This study investigates the expression, the inflammatory, and pro-thrombotic effects of B1R in a model of septic shock in diabetic rats. Sprague-Dawley rats were made diabetic with streptozotocin (STZ) (65 mg/kg, i.p.). Four days later, control and STZ-diabetic rats were injected with lipopolysaccharide (LPS) (2 mg/kg, i.p.) or the vehicle. B1R antagonist (SSR240612, 10 mg/kg by gavage) was given either acutely (12 and 24 h prior to endpoint analysis) or daily for up to 7 days. Moreover, a 7-day treatment was given either with cyclooxygenase (COX)-2 inhibitor (niflumic acid, 5 mg/kg, i.p.), non-selective COX-1 and COX-2 inhibitor (indomethacin, 10 mg/kg, i.p.), non-selective nitric oxide synthase (NOS) inhibitor (L-NAME, 50 mg/kg by gavage), iNOS inhibitor (1400W, 5 mg/kg, i.p.), or heparin (100 IU/kg, s.c.). The following endpoints were measured: edema and vascular permeability (Evans blue dye), B1R expression (qRT-PCR, western blot, flow cytometry), aggregation in platelet-rich plasma (optical aggregometry), and organ damage (histology). Rats treated with STZ, LPS, and STZ plus LPS showed significant increases in edema and vascular permeability (heart, kidney, lung, and liver) and increased expression of B1R in heart and kidney (mRNA) and platelets (protein). Lethal septic shock induced by LPS was enhanced in STZ-diabetic rats and was associated with lung and kidney damage, including platelet micro-aggregate formation. SSR240612 prevented all these abnormalities as well as STZ-induced hyperglycemia and LPS-induced hyperthermia. Similarly to SSR240612, blockade of iNOS and COX-2 improved survival. Data provide the first evidence that kinin B1R plays a primary role in lethal thrombosis in a rat model of septic shock in diabetes. Pharmacological rescue was made possible with B1R antagonism or by inhibition of iNOS and COX-2, which may act as downstream mechanisms.

Changes in blood aggregation with differences in molecular weight and degree of deacetylation of chitosan

Because the molecular weight (Mw) and degree of deacetylation (DDA) of chitosan can vary depending on the purification method, the identification of appropriate chitosan structures is important for developing more effective hemostatic agents. In this study, the influence of varying Mw and DDA of chitosan on blood aggregation was characterized by 10 types of chitosan with different Mw and DDA, including oligomers. The highest aggregation of whole blood, washed erythrocytes and platelets in platelet-rich plasma (PRP) were observed in chitosan with Mw of 8.6 kDa or more and with DDA of 75 to 88%. However, chitosan with too high Mw (247 kDa) inhibited the aggregation of whole blood, washed erythrocytes and PRP at high chitosan concentration. At certain concentrations, chitosan with 75–85% DDA and 50–190 kDa and chitosan with 87.6% DDA and 247 kDa both aggregated proteins in PRP. Chitosan oligomer did not affect blood aggregation. The results suggested that the aggregation by chitosan depended on the interaction of positively charged chitosan with negatively charged erythrocytes, platelets and plasma protein. It seemed that a suitable balance of positive charge in chitosan and negative charge in hemocytes and some kinds of proteins was important. To develop a hemostatic with effective blood aggregation, the chitosan should not be limited to a single Mw and a single DDA but may be a mixed chitosan with wide range of Mw (8.6–247 kDa) and DDA of 75 to 88%.

Anticoagulant mechanism and platelet deaggregation property of a non-cytotoxic, acidic phospholipase A2 purified from Indian cobra (Naja naja) venom: inhibition of anticoagulant activity by low molecular weight heparin.

In the present study, anticoagulant and platelet modulating activities of an acidic phospholipase A2 (NnPLA2-I) purified from Indian cobra Naja naja venom was investigated. The NnPLA2-I displayed a mass of 15.2 kDa and 14,186.0 Da when analyzed by SDS-PAGE and MALDI-TOF-MS, respectively. Peptide mass fingerprinting analysis of the NnPLA2-I showed its significant similarity with phospholipase A2 enzymes purified from cobra venom. BLAST analysis of one tryptic peptide sequence of NnPLA2-I demonstrated putative conserved domains of the PLA2-like superfamily. The Km and Vmax values of NnPLA2-I toward hydrolysis of its most preferred substrate-phosphotidylcholine (PC)-were determined to be 0.72 mM and 29.3 μmol min(-1) mg(-1), respectively. The anticoagulant activity of NnPLA2-I was found to be higher than the anticoagulant activity of heparin/AT-III or warfarin. The histidine modifying reagent, monovalent and polyvalent antivenom differentially inhibited the catalytic and anticoagulant activities of NnPLA2-I. Low molecular weight heparin did not inhibit the catalytic and platelet deaggregation activity of NnPLA2-I, albeit its anticoagulant activity was significantly reduced. The NnPLA2-I showed a non-enzymatic, mixed inhibition of thrombin with a Ki value of 9.3 nM. Heparin significantly decreased, with an IC50 value of 15.23 mIU, the thrombin inhibitory activity of NnPLA2-I. The NnPLA2-I uniquely increased the amidolytic activity of FXa without influencing its prothrombin activating property. NnPLA2-I showed dose-dependent deaggregation of platelet rich plasma (PRP) and inhibited the collagen and thrombin-induced aggregation of PRP. However, deaggregation of washed platelets by NnPLA2-I demonstrated in presence of PC or platelet poor plasma. Alkylation of histidine residue of NnPLA2-I resulted in 95% and 21% reduction of its platelet deaggregation and platelet binding properties, respectively. NnPLA2-I did not show cytotoxicity against human glioblastoma U87MG cells, bactericidal or hemolytic activity. The future therapeutic application of NnPLA2-I for treatment and prevention of cardiovascular disorders is therefore suggested.