Thrombin-induced Platelet Aggregation Research Articles

Abstract 18972: Safety and Efficacy of Vorapaxar in Secondary Prevention of Atherosclerotic Disease: A Meta-analysis of Randomized Control Trials

Background: A substantial risk of residual cardiovascular events remains despite the current standard antiplatelet therapy in patients with atherosclerotic disease. Protease activating receptor (PAR-1) antagonist Vorapaxar is a novel antiplatelet agent acting by inhibition of thrombin-induced platelet aggregation. Recently, FDA has approved vorapaxar for long term secondary prevention of vascular events in stable patients with a prior MI or PAD, and without history of stroke or transient ischemic attack. However, these recommendations are largely based on a single phase 3 randomized control trial (TRA 2P TIMI-50). Objective: To study the cumulative evidence for vorapaxar use in patients with atherosclerotic cardiovascular disease. Methods: A systematic review of randomized control trials in MEDLINE, EMBASE, EBSCO, CINAHL, Web of Science and Cochrane databases comparing vorapaxar with placebo was performed. Pre-specified efficacy endpoints were all-cause mortality, CV mortality, myocardial infarction, stroke and repeat revascularization. The pre-specified safety endpoint was the composite of TIMI major and minor bleeding. Risk ratios were used as the metric of choice by applying random effects models.. Results: A total of 5 RCTs with 40,630 patients were included. Compared with placebo, vorapaxar led to a significant risk reduction in terms of myocardial infarction [RR 0.93; 95% CI 0.80-0.92] and stroke [RR 0.84; 95% CI 0.72-0.97]. No differences were observed between vorapaxar and placebo with respect to all-cause mortality [RR 0.99; 95% CI 0.90-1.07], cardiovascular mortality [RR 0.93; 95% CI 0.84-1.02], and repeat revascularization [RR 0.96; 95% CI 0.85-1.08]. Vorapaxar was associated with a higher risk of TIMI major and minor bleeding [RR 1.30; 95% CI 1.02-1.65] compared with placebo. There was insignificant (I-squared) heterogeneity between studies. Conclusion: Vorapaxar reduces the risk of ischemic events in patients with atherosclerotic disease at the cost of an increased bleeding risk.

Abstract 19967: Differential Roles of the NADPH-Oxidase 1 and 2 in Platelet Activation and Thrombosis

Objective: Reactive oxygen species (ROS) generated from activated platelets is known to regulate platelet activation. However, it remains unclear whether and how different isoforms of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases (NOXs) play roles in different platelet activation pathways. Here we investigated the role of NOX1 and NOX2 in different platelet activation pathways using NOX1 and NOX2 knockout mice. Approach and Results: NOX1-/- platelets showed selective defects in G protein coupled receptor (GPCR)-mediated platelet activation induced by thrombin, protease-activated receptor 4 agonist peptide (PAR4AP) and thromboxane A2 analog U46619, but was not affected in platelet activation induced by collagen-related peptide (CRP), a glycoprotein VI (GPVI) agonist. In contrast, NOX2-/- platelets showed potent inhibition of CRP-induced platelet activation, and also showed partial inhibition of thrombin-induced platelet aggregation and secretion. Consistently, production of reactive oxygen species (ROS) was inhibited in NOX1-/- platelets stimulated with thrombin, but not CRP, whereas NOX2-/- platelets showed reduced ROS generation induced by CRP or thrombin. Interestingly, laser-induced arterial thrombosis was impaired in NOX2-/- mice, and in thrombocytopenic mice transfused with NOX2-/- platelets, suggesting an important role for NOX2-dependent platelet ROS production in the laser-induced injury model of thrombosis. Conclusions: NOX1 and NOX2 play differential roles in different platelet activation pathways: NOX1 mediates GPCR-mediated ROS production and platelet activation, whereas NOX2 plays a general role in GPVI- and GPCR-induced ROS production and platelet activation in vitro , and in laser-induced thrombosis in vivo .

Cartilage oligomeric matrix protein is a natural inhibitor of thrombin

AbstractThrombin is an effector enzyme for hemostasis and thrombosis; however, endogenous regulators of thrombin remain elusive. Cartilage oligomeric matrix protein (COMP), a matricellular protein also known as thrombospondin-5, is essential for maintaining vascular homeostasis. Here, we asked whether COMP is involved in the process of blood coagulation. COMP deficiency shortened tail-bleeding and clotting time and accelerated ferric-chloride–induced thrombosis in mice. The absence of COMP had no effect on platelet count. In contrast, COMP specifically inhibited thrombin-induced platelet aggregation, activation, and retraction and the thrombin-mediated cleavage of fibrinogen. Furthermore, surface plasmon resonance analysis revealed direct thrombin-COMP binding (KD = 1.38 ± 0.24 μM). In particular, blockage of thrombin exosites with compounds specific for exosite I (hirudin and HD1 aptamer) or exosite II (heparin and HD22 aptamer) impaired the COMP-thrombin interaction, indicating a 2-site binding mechanism. Additionally, epidermal growth factor–like repeats (amino acids 84-261) were identified as a COMP binding site for thrombin. Moreover, COMP was expressed in and secreted by platelets. Using bone marrow transplantation and platelet transfusion to create chimeric mice, platelet-derived but not vessel-wall–derived COMP was demonstrated to inhibit coagulation. Taken together, COMP is an endogenous thrombin inhibitor and negative regulator of hemostasis and thrombosis.

HMGB1: a novel protein that induced platelets active and aggregation via Toll-like receptor-4, NF-κB and cGMP dependent mechanisms.

BackgroundThrombotic diseases are a group of prevalent and life-threatening diseases. Selective inhibition of pathological thrombosis holds the key to treat variety of thrombotic diseases. The pathological thrombosis can be induced by either tissue necrosis and deregulated inflammation. HMGB1, as an important proinflammatory cytokine and a late mediator, also involves on thrombosis disease. However, the underlying mechanisms are not fully understood.MethodsImmunofluorescence, ELISA assay, Platelet Aggregation, Thromboelastogram (TEG) analyzes. Flow cytometric analysis and Western blot analysis were used to investigated the role of HMGB1 in platelet aggregation and obtained following observations.ResultsBy doing so, we obtained the following observations: i) Highly purified HMGB1 recombinant protein induces platelet aggregation and secretion in a dose-dependent manner in the presence of serum. ii) Low concentration of extracellular HMGB1 could synergistically promote subthreshold concentration of collagen or thrombin induced platelet aggregation. iii) Extracellular HMGB1 promoted platelet aggregation in a platelet-expressed GPIIb/IIIa-dependent manner. iv) We proposed that extracellular HMGB1 seems to promote the phosphorylation of GPIIb/IIIa and subsequent platelet aggregation via TLR4/NF-κB and cGMP pathway.ConclusionsIn this study, we provide evidence for the hypothesis that HMGB1 interact with platelet might play an important role in the haemostasis and thrombotic diseases. Our research might be provide an interesting avenue for the treatment of thrombotic diseases in the future.

Inhibitory Effects of Rice Bran Water Extract Fermented Lactobacillus plantarum due to cAMP-dependent Phosphorylation of VASP (Ser157) on human Platelet Aggregation

In this study, we investigated the effect of rice bran water extract fermented with Lactobacillus plantarum KCCM-12116 (RBLp) on ADP (20 μM)-, collagen (10 μg/mL)-, and thrombin (0.2 U/mL)-stimulated platelet aggregation. RBLp dose-dependently inhibited ADP-, collagen-, and thrombin-induced platelet aggregation, with IC50 values of 501.1, 637.2, and > 2,000 μg/mL, respectively. The platelet aggregation induced by ADP plus RBLp (750 μg/mL) was increased by the adenylate cyclase inhibitor, SQ22536, and the cAMP-dependent protein kinase (A-kinase) inhibitor, Rp-8-Br-cAMPS. Treatment with RBLp increased the phosphorylation of VASP (Ser 157 ), an A-kinase substrate, which was also inhibited by SQ22536 and Rp-8-Br-cAMPS. It is thought that the RBLp-induced increases in cAMP contributed to the phosphorylation of VASP (Ser 157 ), which in turn resulted in an inhibition of ADP-induced platelet aggregation, thereby indicating that RBLp has an antiplatelet effect via cAMP-dependent phosphorylation of VASP (Ser 157 ). Thus, RBLp may have therapeutic potential for the treatment (or prevention) of platelet aggregation-mediated diseases, such as thrombosis, myocardial infarction, atherosclerosis, and ischemic cerebrovascular disease.

Autoantibodies to Factor XII and Kininogen-Dependent Antiphosphatidylethanolamine Antibodies in Patients with Recurrent Pregnancy Loss Augment Platelet Aggregation.

Numerous studies have suggested that factor XII (FXII) deficiency, autoantibodies to FXII (anti-FXII), and antiphosphatidylethanolamine antibodies (aPE) are associated with recurrent pregnancy loss (RPL). aPE in RPL patients recognize the LDC27 peptide of kininogen domain 3. Anti-FXII in RPL patients recognizes the heavy chain of FXII, especially the amino-terminal sequences IPP30 peptide. Previous studies suggested that LDC27 and IPP30 are the responsible sites competing for the same binding site on platelets and inhibiting augmentation of thrombin-induced platelet aggregation. Our aim was to study the influence of antibodies to LDC27 and IPP30 on platelet aggregation. In fifteen healthy volunteers, platelet aggregation induced by γ-thrombin in the presence or absence of antibodies to LDC27 and IPP30 was measured. Sixteen RPL patients who were positive for anti-FXII were measured for spontaneous small platelet aggregate (SSPA) formation. Antibodies to LDC27 and IPP30 markedly increased aggregation of normal platelets stimulated by γ-thrombin. Augmentation of SSPA formation was more frequent in the patients with RPL who were positive for anti-FXII than in the control group (P = 0.003). This study strongly supports the hypothesis that aPE and anti-FXII may cause RPL due to disruption of the normal antithrombotic effects of kininogens and FXII.

Total saponin from Korean Red Ginseng inhibits binding of adhesive proteins to glycoprotein IIb/IIIa via phosphorylation of VASP (Ser157) and dephosphorylation of PI3K and Akt

BackgroundBinding of adhesive proteins (i.e., fibrinogen, fibronectin, vitronectin) to platelet integrin glycoprotein IIb/IIIa (αIIb/β3) by various agonists (thrombin, collagen, adenosine diphosphate) involve in strength of thrombus. This study was carried out to evaluate the antiplatelet effect of total saponin from Korean Red Ginseng (KRG-TS) by investigating whether KRG-TS inhibits thrombin-induced binding of fibrinogen and fibronectin to αIIb/β3. MethodsWe investigated the effect of KRG-TS on phosphorylation of vasodilator-stimulated phosphoprotein (VASP) and dephosphorylation of phosphatidylinositol 3-kinase (PI3K) and Akt, affecting binding of fibrinogen and fibronectin to αIIb/β3, and clot retraction. ResultsKRG-TS had an antiplatelet effect by inhibiting the binding of fibrinogen and fibronectin to αIIb/β3 via phosphorylation of VASP (Ser157), and dephosphorylation of PI3K and Akt on thrombin-induced platelet aggregation. Moreover, A-kinase inhibitor Rp-8-Br-cyclic adenosine monophosphates (cAMPs) reduced KRG-TS-increased VASP (Ser157) phosphorylation, and increased KRG-TS-inhibited fibrinogen-, and fibronectin-binding to αIIb/β3. These findings indicate that KRG-TS interferes with the binding of fibrinogen and fibronectin to αIIb/β3 via cAMP-dependent phosphorylation of VASP (Ser157). In addition, KRG-TS decreased the rate of clot retraction, reflecting inhibition of αIIb/β3 activation. In this study, we clarified ginsenoside Ro (G-Ro) in KRG-TS inhibited thrombin-induced platelet aggregation via both inhibition of [Ca2+]i mobilization and increase of cAMP production. ConclusionThese results strongly indicate that KRG-TS is a beneficial herbal substance inhibiting fibrinogen-, and fibronectin-binding to αIIb/β3, and clot retraction, and may prevent platelet αIIb/β3-mediated thrombotic disease. In addition, we demonstrate that G-Ro is a novel compound with antiplatelet characteristics of KRG-TS.

Mitochondria-Derived Reactive Oxygen Species Play an Important Role in Doxorubicin-Induced Platelet Apoptosis

Doxorubicin (DOX) is an effective chemotherapeutic agent; however; its use is limited by some side effects; such as cardiotoxicity and thrombocytopenia. DOX-induced cardiotoxicity has been intensively investigated; however; DOX-induced thrombocytopenia has not been clearly elucidated. Here we show that DOX-induced mitochondria-mediated intrinsic apoptosis and glycoprotein (GP)Ibα shedding in platelets. DOX did not induce platelet activation; whereas; DOX obviously reduced adenosine diphosphate (ADP)- and thrombin-induced platelet aggregation; and impaired platelet adhesion on the von Willebrand factor (vWF) surface. In addition; we also show that DOX induced intracellular reactive oxygen species (ROS) production and mitochondrial ROS generation in a dose-dependent manner. The mitochondria-targeted ROS scavenger Mito-TEMPO blocked intracellular ROS and mitochondrial ROS generation. Furthermore; Mito-TEMPO reduced DOX-induced platelet apoptosis and GPIbα shedding. These data indicate that DOX induces platelet apoptosis; and impairs platelet function. Mitochondrial ROS play a pivotal role in DOX-induced platelet apoptosis and GPIbα shedding. Therefore; DOX-induced platelet apoptosis might contribute to DOX-triggered thrombocytopenia; and mitochondria-targeted ROS scavenger would have potential clinical utility in platelet-associated disorders involving mitochondrial oxidative damage.

Design, synthesis and structural exploration of novel fluorinated dabigatran derivatives as direct thrombin inhibitors

Twenty-one fluorinated dabigatran derivatives were designed based on the bioisosteric principle. All derivatives were synthesised and evaluated for their thrombin inhibitory activity in vitro. Among these compounds, 14h, 14m, 14s and 14t were potent and the activity was in the range of reference drug, dabigatran. Three structural changes were introduced in these 21 compounds to elucidate the structure–activity relationship of the drugs. In addition, prodrugs of compounds 14h and 14s were developed to investigate their anticoagulant activities in vivo. In these experiments, compound 16 showed a fairly strong inhibitory effect on thrombin-induced platelet aggregation, and demonstrated potent activity for inhibiting arteriovenous thrombosis with an inhibition rate of (73 ± 6) %, which was comparable to that of dabigatran etexilate (76 ± 2) %. Moreover, molecular docking studies were performed to understand the binding interactions of active compounds 14h, 14s and 14t with thrombin protein (PDB ID:1KTS). Contour maps obtained from the 3D-QSAR model are meaningful in designing more active molecules to act as direct inhibitors of thrombin.

Inhibitory effects of total saponin from Korean Red Ginseng on [Ca2+]i mobilization through phosphorylation of cyclic adenosine monophosphate-dependent protein kinase catalytic subunit and inositol 1,4,5-trisphosphate receptor type I in human platelets

BackgroundIntracellular Ca2+([Ca2+]i) is a platelet aggregation-inducing molecule. Therefore, understanding the inhibitory mechanism of [Ca2+]i mobilization is very important to evaluate the antiplatelet effect of a substance. This study was carried out to understand the Ca2+-antagonistic effect of total saponin from Korean Red Ginseng (KRG-TS). MethodsWe investigated the Ca2+-antagonistic effect of KRG-TS on cyclic nucleotides-associated phosphorylation of inositol 1,4,5-trisphosphate receptor type I (IP3RI) and cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) in thrombin (0.05 U/mL)-stimulated human platelet aggregation. ResultsThe inhibition of [Ca2+]i mobilization by KRG-TS was increased by a PKA inhibitor (Rp-8-Br-cAMPS), which was more stronger than the inhibition by a cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) inhibitor (Rp-8-Br-cGMPS). In addition, Rp-8-Br-cAMPS inhibited phosphorylation of PKA catalytic subunit (PKAc) (Thr197) by KRG-TS. The phosphorylation of IP3RI (Ser1756) by KRG-TS was very strongly inhibited by Rp-8-Br-cAMPS compared with that by Rp-8-Br-cGMPS. These results suggest that the inhibitory effect of [Ca2+]i mobilization by KRG-TS is more strongly dependent on a cAMP/PKA pathway than a cGMP/PKG pathway. KRG-TS also inhibited the release of adenosine triphosphate and serotonin. In addition, only G-Rg3 of protopanaxadiol in KRG-TS inhibited thrombin-induced platelet aggregation. ConclusionThese results strongly indicate that KRG-TS is a potent beneficial compound that inhibits [Ca2+]i mobilization in thrombin–platelet interactions, which may result in the prevention of platelet aggregation-mediated thrombotic disease.

Inhibitory Effects of Cytosolic Ca(2+) Concentration by Ginsenoside Ro Are Dependent on Phosphorylation of IP3RI and Dephosphorylation of ERK in Human Platelets.

Intracellular Ca2+ ([Ca2+]i) is platelet aggregation-inducing molecule and is involved in activation of aggregation associated molecules. This study was carried out to understand the Ca2+-antagonistic effect of ginsenoside Ro (G-Ro), an oleanane-type saponin in Panax ginseng. G-Ro, without affecting leakage of lactate dehydrogenase, dose-dependently inhibited thrombin-induced platelet aggregation, and the half maximal inhibitory concentration was approximately 155 μM. G-Ro inhibited strongly thrombin-elevated [Ca2+]i, which was strongly increased by A-kinase inhibitor Rp-8-Br-cAMPS compared to G-kinase inhibitor Rp-8-Br-cGMPS. G-Ro increased the level of cAMP and subsequently elevated the phosphorylation of inositol 1, 4, 5-triphosphate receptor I (IP3RI) (Ser1756) to inhibit [Ca2+]i mobilization in thrombin-induced platelet aggregation. Phosphorylation of IP3RI (Ser1756) by G-Ro was decreased by PKA inhibitor Rp-8-Br-cAMPS. In addition, G-Ro inhibited thrombin-induced phosphorylation of ERK 2 (42 kDa), indicating inhibition of Ca2+ influx across plasma membrane. We demonstrate that G-Ro upregulates cAMP-dependent IP3RI (Ser1756) phosphorylation and downregulates phosphorylation of ERK 2 (42 kDa) to decrease thrombin-elevated [Ca2+]i, which contributes to inhibition of ATP and serotonin release, and p-selectin expression. These results indicate that G-Ro in Panax ginseng is a beneficial novel Ca2+-antagonistic compound and may prevent platelet aggregation-mediated thrombotic disease.

Loss of Autophagy Leads to Megakaryocytes Differentiation Failure and Defective Platelets Function

BackgroundMegakaryocytes (MKs), large progenitor cells residing in the bone marrow, are the source of platelets. In 2009, Colosetti P et al. reported that PMA and SB could induce megakaryocytic differentiation of the chronic myelogenous leukemia cell line K562 by triggering autophagy. It gives us the first insight of autophagy induction in in vitro megakaryocytic differentiation. Although Feng W et al. reported autophagy also exists in human platelets, the role of autophagy in megakaryocyte-platelet commitment axis remains poorly understood. In this study, we elucidated the biological effects of autophagy deficiency on megakaryopoiesis and thrombosis using hematopoietic system conditionally atg7 knockout mice.Methods and MaterialsTo evaluate the biological effects of autophagy deficiency on platelets, the following experiments were performed: (1) complete blood count of wild type and atg7-/- mice, (2) the tail bleeding time assay of wild type and Atg7-/- mice, (3) the effect of atg7 knockout on platelet aggregation and activation of CD62P and JON/A (αIIbβ3) were analyzed by flow cytometry. To assess whether the observed changes in platelets of atg7-/- mice result in aberrations of megakaryopoiesis, the following experiments were performed: (1) the percentage of BM CD41+CD61+ cells was analyzed by flow cytometry, (2) the AchE activity assay of platelets and murine BM Lin- cells cultured with murine TPO and SCF, (3) Morphology of megakaryocytes derived from BM Lin- cells was evaluated by Wright-Giemsa staining, (4) megakaryocytic differentiation from BM Lin- cells was evaluated by CD41/forward-scatter (FSC) dot plot. To evaluate the role of reactive oxygen species in MK differentiation, the Lin- cells were stained with MitoTracker Green and MitoSox Red and then analyzed by flow cytometry.Results(1) The number of platelets in the peripheral blood of atg7-/- mice was significantly decreased (WT: 904.2±75.5, Atg7+/-: 942.8±136.3, Atg7-/-: 330.5±282.2, p<0.01), while the size of platelets (MPV) was increased compared with WT mice (WT: 5.6±0.1, Atg7+/-: 5.7±0.1, atg7-/-: 6.8±0.5, p<0.01). (2) The bleeding time was significantly longer (WT: 51.5±14.8s, Atg7-/-: 915.2±282.9s, p<0.01) and thrombin-induced platelet aggregation was decreased (WT: 97.5±2.5, Atg7+/-: 62.5±7.5, Atg7-/-: 7.75±7.25, p<0.05) in Atg7-/- mice than in wild-type mice. (3) The activation of CD62P (WT: 14.5±0.09, atg7+/-: 11.17±0.06, atg7-/-: 3.2±0.03, p<0.01) and JON/A (αIIbβ3) (WT: 48.1±0.1, atg7+/-: 13.5±0.1, atg7-/-: 5.9±0.2, p<0.01) was decreased in atg7-/- platelets. These results indicated that atg7-dependent autophagy is important for thrombosis and platelet function. (4) The percentage of CD41+CD61+ cells was decreased in bone marrow of atg7-/- mice (WT: 30.4±0.6, atg7+/-: 27.9±1.3, atg7-/-: 18.9±0.3, p<0.01). (5) In mice lacking autophagy, both the Lin- cells stimulated by TPO (WT: 0.35±0.03, atg7-/-: 0.22±0.05, p<0.01) and the platelets collected through the inferior vena cava (WT: 0.099±0.005, atg7-/-: 0.05±0.009, p<0.01) had significantly lower AChE activity compared with WT mice. (6) Low level of CD41+/FSChigh cells were seen in the in vitro culture of atg7-/- BM Lin- cells with TPO and SCF ( WT:5.6±2.2, Atg7-/-: 0.2±0.03, p<0.01). These results reflected a significant reduction in MK differentiation from autophagy defective hematopoietic progenitors. An accumulation of mitochondria (WT: 547.3±7.0, atg7-/-: 737.8±126.6, p<0.01) and mitochondrial superoxide (WT: 280.2±4.8, atg7-/-: 343.8±42.4, p<0.05) was found in atg7-/- BM Lin- cells, which may severely disturb the progress of MK differentiation.ConclusionAutophagy is essential for the megakaryopoiesis and thrombosis by maintaining mitochondrial homeostasis. Elevated reactive oxygen species might be the cause of megakaryocytic differentiation blockade. DisclosuresNo relevant conflicts of interest to declare.

Lovastatin Induces Platelet Apoptosis

IntroductionLovastatin, the inhibitors of 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase, is widely used in treatments of coronary artery diseases. Lovastatin induces apoptosis in nucleate cells, such as macrophages, cancer cells, and skeletal muscle cells. Lovastatin has side effects of thrombocytopenia and hemorrhage, however, the possible pathogenesis of the side effects have still remained unknown.Methods and ResultsIn this study, we found that collagen and thrombin-induced platelet aggregation was obviously reduced in platelets treated by lovastatin, while platelet activation (expression of P-selectin or PAC-1 binding on platelet surface) was not detected. The apoptosis markers in both intrinsic mitochondrial pathway (ΔΨm changes, PS exposure, Bax/Bak and Bcl-XL expression, and caspase9/3 activation) and extrinsic pathway (caspase 8) were detected in platelets treated with lovastatin. Apoptosis decreased in platelets after being pretreated by tetrapeptide arginine-glycine-aspartic acid-serine (RGDS) peptide (the GPIIb/IIIa antagonist). Lovastatin wasintraperitoneally injected into C57 mice at a dose of 5 mg/kg for four hours. The circulating platelets were significantly decreased by lovastatin compared with that of vehicle control.ConclusionLovastatin induced platelet apoptosis by both intrinsic mitochondrial and extrinsic pathways, which might be the possible pathogenesis of thrombocytopenia or hemorrhage in patients treated with lovastatin. DisclosuresNo relevant conflicts of interest to declare.

The effects of magnesium, acetylsalicylic acid, and emoxypine on platelet aggregation

This paper summarizes the results of investigation of thrombin-induced platelet aggregation in the absence and presence of magnesium sulfate, acetylsalicylic acid, and emoxypine. Magnesium sulfate, as well as acetylsalicylic acid and emoxypine, were shown to reduce the degree of platelet aggregation when used individually, while the combined action of these three substances did not have a similar effect. The data obtained can be used for selection of ischemic stroke treatment strategies.

Thymidine phosphorylase participates in platelet signaling and promotes thrombosis.

Platelets contain abundant thymidine phosphorylase (TYMP), which is highly expressed in diseases with high risk of thrombosis, such as atherosclerosis and type II diabetes mellitus. To test the hypothesis that TYMP participates in platelet signaling and promotes thrombosis. By using a ferric chloride (FeCl3)-induced carotid artery injury thrombosis model, we found time to blood flow cessation was significantly prolonged in Tymp(-/-) and Tymp(+/-) mice compared with wild-type mice. Bone marrow transplantation and platelet transfusion studies demonstrated that platelet TYMP was responsible for the antithrombotic phenomenon in the TYMP-deficient mice. Collagen-, collagen-related peptide-, adenosine diphosphate-, or thrombin-induced platelet aggregation were significantly attenuated in Tymp(+/-) and Tymp(-/-) platelets, and in wild type or human platelets pretreated with TYMP inhibitor KIN59. Tymp deficiency also significantly decreased agonist-induced P-selectin expression. TYMP contains an N-terminal SH3 domain-binding proline-rich motif and forms a complex with the tyrosine kinases Lyn, Fyn, and Yes in platelets. TYMP-associated Lyn was inactive in resting platelets, and TYMP trapped and diminished active Lyn after collagen stimulation. Tymp/Lyn double haploinsufficiency diminished the antithrombotic phenotype of Tymp(+/-) mice. TYMP deletion or inhibition of TYMP with KIN59 dramatically increased platelet-endothelial cell adhesion molecule 1 tyrosine phosphorylation and diminished collagen-related peptide- or collagen-induced AKT phosphorylation. In vivo administration of KIN59 significantly inhibited FeCl3-induced carotid artery thrombosis without affecting hemostasis. TYMP participates in multiple platelet signaling pathways and regulates platelet activation and thrombosis. Targeting TYMP might be a novel antiplatelet and antithrombosis therapy.

Overview of the 2014 Food and Drug Administration Cardiovascular and Renal Drugs Advisory Committee meeting about vorapaxar.

Patients with a history of heart disease are at increased risk for future cardiovascular events, including myocardial infarction (MI), stroke, and death resulting from cardiovascular disease. Although coronary artery disease remains the leading cause of mortality worldwide, advancements in medical therapy, particularly antithrombotic agents, have improved patient outcomes.1–4 Although the trend for MI and death after acute coronary syndrome (ACS) over the past 20 years has declined,5,6 such medical advancements are still met with discouraging rates of death and MI after revascularization.7 To this end, it is the recommendation of the American College of Cardiology and American Heart Association that all ACS patients, regardless of whether percutaneous coronary intervention was performed, be treated with dual antiplatelet therapy (DAPT) for 1 year ( Class IA ).8 Such recommendations are based largely on results from the Clopidogrel in Unstable Angina to Prevent Recurrent Events (CURE) trial, which assessed aspirin versus DAPT with aspirin and clopidogrel for the reduction of ischemic end points in patients presenting with non–ST-segment–elevation ACS who did not receive percutaneous coronary intervention.9 Seeing a need for further risk reduction in long-term management of patients with established atherothrombotic risk, the Thrombin Receptor Antagonist in Secondary Prevention of Atherothrombotic Ischemic Events (TRA 2P)–Thrombolysis in Myocardial Infarction (TIMI) 50 was conducted. The study drug, vorapaxar (Zontivity, Merck, NJ), is an antagonist of the platelet protease-activated receptor (PAR-1) and inhibits thrombin-induced platelet aggregation.10,11 Vorapaxar was shown to have a significant benefit for the reduction of cardiovascular death and ischemic complications in addition to secondary prevention.12 Vorapaxar was also tested in the Thrombin Receptor Antagonist for Clinical Event Reduction in Acute Coronary Syndrome (TRACER) trial as part of adjunct therapy in the non–ST-segment–elevation ACS population.13 In this population, however, …

In vitro effects of tacrolimus on platelet function

To investigate the in vitro effects of immune inhibitor tacrolimus on platelet function. Fresh venous blood was collected from healthy volunteers at ages of 18-25 years old, who are not taking antiplatelet drugs within two weeks. The platelets were isolated from the blood and incubated with different concentrations of tacrolimus (0.06, 0.6, 6, 60, 120, 240 μmol/L) at 37 °C for 2 hours, and then the changes of mitochondrial membrane potential and P-selection of platelets were detected by flow cytometry, the expression of apoptosis related protein by Western Blot, and the change of the platelet aggregation function by platelet aggregation analyzer. Tacrolimus at concentration of 0.06 μmol/L could promote collagen induced platelet aggregation, inhibit thrombin induced platelet aggregation, have no effect on ristocetin and vWF induced platelet aggregation function. Tacrolimus at concentration of 120 μmol/L and 240 μmol/L could reduce the platelet mitochondrial membrane potential and induce the expression of apoptosis protein caspase-3. In vitro experimental results showed that high concentration of tacrolimus could lead to platelet apoptosis. But the current therapeutic dose of tacrolimus at 0.06 μmol/L (which is equivalent to 50 ng/ml blood concentration) could have different effects on platelet aggregation function according to different stimulating agents.

Protease-activated receptor 4: a critical participator in inflammatory response.

Protease-activated receptors (PARs) are G protein-coupled receptors of which four members PAR1, PAR2, PAR3, and PAR4 have been identified, characterized by a typical mechanism of activation involving various related proteases. The amino-terminal sequence of PARs is cleaved by a broad array of proteases, leading to specific proteolytic cleavage which forms endogenous tethered ligands to induce agonist-biased PAR activation. The biological effect of PARs activated by coagulation proteases to regulate hemostasis and thrombosis plays an enormous role in the cardiovascular system, while PAR4 can also be activated by trypsin, cathepsin G, the activated factor X of the coagulation cascade, and trypsin IV. Irrespective of its role in thrombin-induced platelet aggregation, PAR4 activation is believed to be involved in inflammatory lesions, as show by investigations that have unmasked the effects of PAR4 on neutrophil recruitment, the regulation of edema, and plasma extravasation. This review summarizes the roles of PAR4 in coagulation and other extracellular protease pathways, which activate PAR4 to participate in normal regulation and disease.

Two acidic, anticoagulant PLA2 isoenzymes purified from the venom of monocled cobra Naja kaouthia exhibit different potency to inhibit thrombin and factor Xa via phospholipids independent, non-enzymatic mechanism.

BackgroundThe monocled cobra (Naja kaouthia) is responsible for snakebite fatality in Indian subcontinent and in south-western China. Phospholipase A2 (PLA2; EC 3.1.1.4) is one of the toxic components of snake venom. The present study explores the mechanism and rationale(s) for the differences in anticoagulant potency of two acidic PLA2 isoenzymes, Nk-PLA2α (13463.91 Da) and Nk-PLA2β (13282.38 Da) purified from the venom of N. kaouthia.Principal FindingsBy LC-MS/MS analysis, these PLA2s showed highest similarity (23.5% sequence coverage) with PLA2 III isolated from monocled cobra venom. The catalytic activity of Nk-PLA2β exceeds that of Nk-PLA2α. Heparin differentially regulated the catalytic and anticoagulant activities of these Nk-PLA2 isoenzymes. The anticoagulant potency of Nk-PLA2α was comparable to commercial anticoagulants warfarin, and heparin/antithrombin-III albeit Nk-PLA2β demonstrated highest anticoagulant activity. The anticoagulant action of these PLA2s was partially contributed by a small but specific hydrolysis of plasma phospholipids. The strong anticoagulant effect of Nk-PLA2α and Nk-PLA2β was achieved via preferential, non-enzymatic inhibition of FXa (Ki = 43 nM) and thrombin (Ki = 8.3 nM), respectively. Kinetics study suggests that the Nk-PLA2 isoenzymes inhibit their “pharmacological target(s)” by uncompetitive mechanism without the requirement of phospholipids/Ca2+. The anticoagulant potency of Nk-PLA2β which is higher than that of Nk-PLA2α is corroborated by its superior catalytic activity, its higher capacity for binding to phosphatidylcholine, and its greater strength of thrombin inhibition. These PLA2 isoenzymes thus have evolved to affect haemostasis by different mechanisms. The Nk-PLA2β partially inhibited the thrombin-induced aggregation of mammalian platelets suggesting its therapeutic application in the prevention of unwanted clot formation.Conclusion/SignificanceIn order to develop peptide-based superior anticoagulant therapeutics, future application of Nk-PLA2α and Nk-PLA2β for the treatment and/or prevention of cardiovascular disorders are proposed.

Targeting the anionic region of human protease‐activated receptor 4 inhibits platelet aggregation and thrombosis without interfering with hemostasis

Human platelet activation and aggregation is a complex process. To date, many therapies have been developed targeting proteins that mediate this process to prevent unwanted activation. However, the current standard of care for acute coronary syndromes still has limitations, including bleeding risk. To evaluate the protease-activated receptor 4 (PAR4) anionic cluster as a viable antiplatelet target by using a polyclonal antibody (CAN12). We used western blotting, aggregation and secretion ex vivo to evaluate the ability of CAN12 to interact with PAR4 and inhibit platelet activation. The effects of CAN12 in vivo were evaluated with the Rose Bengal arterial thrombosis model and two models of hemostasis. CAN12 was able to interact with human PAR4 and delay PAR4 cleavage. In addition, CAN12 inhibited thrombin-induced human platelet aggregation and secretion in a dose-dependent manner. The specificity of CAN12 was agonist-dependent. In vivo, we determined that CAN12 was able to inhibit arterial thrombosis, and, using two independent methods, we found that CAN12 did not influence hemostasis. Targeting the extracellular anionic cluster on PAR4 is a viable novel strategy as an antiplatelet therapy.