Antiplatelet mechanism of an herbal mixture prepared from the extracts of <Emphasis Type="Italic">Phyllostachys pubescens</Emphasis> leaves and <Emphasis Type="Italic">Prunus mume</Emphasis> fruits

Background

Bamboo (Phyllostachys pubescens) leaves and Japanese apricot (Mume fructus) fruit are traditionally recognized to be safe herbs broadly used for food and medicinal purposes in Southeast Asia. Our group previously explored their antiplatelet effects. This study was designed to confirm inhibition effects of PM21 (a 2:1 mixture of bamboo leaf extract and Japanese apricot fruit extract) on platelet aggregation and evaluate its potency to use as an herbal remedy to prevent and/or treat the diseases caused by platelet aggregation and thrombus formation.

Methods

Washed platelets were prepared and platelet aggregation was induced by adding 5 μg/mL collagen. Anti-platelet effects of PM21 (75 mg/kg, 150 mg/kg, and 300 mg/kg for ex vivo and in vivo assays, and 50, 100, 200 μg/mL for in vitro assays) were evaluated. In ex vivo assays, PM21 was orally administered to rats daily after overnight fasting for 3 days and blood was collected 1 h after the final treatment. In vivo antithrombotic effect of PM21 was observed from a carrageenan induced mouse tail thrombosis model.

Results

In ex vivo assay, PM21 inhibited platelet aggregation significantly. PM21 showed a strong antithrombotic effect by reducing significantly the length of mouse tail thrombus. PM21 increased intracellular cAMP level and reduced the release of ATP, TXA₂, and serotonin. PM21 also reduced intracellular concentration of calcium ion, fibrinogen binding to integrin α_IIbβ₃, and phosphorylation of ERK2, p38, PLCγ2, and PI3 K.

Conclusions

PM21 showed remarkable inhibitory effects on platelet aggregation and thrombus formation. Its inhibitory function seems to influence on GPVI binding to its ligand and subsequent initiation of a signaling cascade that involves activation of effector proteins and secretion of effector molecules, such as ATP, TXA₂, serotonin, and Ca²⁺. PM21 also appears to exert its anti-platelet effect by deactivation of ERKs activation pathway as well as inhibition of fibrinogen binding to integrin α_IIbβ₃.

     

Transport Limitations of Nitric Oxide Inhibition of Platelet Aggregation under Flow

Nitric oxide (NO) inhibits platelet aggregation at and near the site of a vascular injury by upregulation of cyclic guanosine monophosphate, which reduces the dimerization of the integrin α _IIb β ₃. The magnitude of NO flux from the vessel wall and the NO concentration that is necessary to inhibit platelet aggregation under physiological flow conditions is unknown. In this study, a NO releasing polymer, diazeniumdiolated dibutylhexanediamine, was integrated into a microfluidic flow assay to determine the relationship between NO wall flux and collagen mediated platelet adhesion, activation and aggregation. A NO flux equal to or greater than 2.5 × 10^?10 mol cm^?2 min^?1 was found to abrogate aggregation, but not initial platelet adhesion, on collagen at 200 and 500 s^?1 as effectively as the α _IIb β ₃ antagonist abciximab. The dynamic range of NO fluxes found to induce measurable inhibition of platelet aggregation spanned from 0.33 × 10^?10 to 2.5 × 10^?10 mol cm^?2 min^?1 at 200–500 s^?1. These fluxes correspond to near-wall NO concentrations of 3–90 nM based on a computational model of NO transport. The model predicts that NO concentration in the platelet rich layer near the wall is kinetically limited, while NO penetration into the lumen is mass transfer limited.     

Rac1 is essential for phospholipase C-γ2 activation in platelets

Platelet activation at sites of vascular injury is triggered through different signaling pathways leading to activation of phospholipase (PL) Cβ or PLCγ2. Active PLCs trigger Ca²⁺ mobilization and entry, which is a prerequisite for adhesion, secretion, and thrombus formation. PLCβ isoenzymes are activated downstream of G protein-coupled receptors (GPCRs), whereas PLCγ2 is activated downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors, such as the major platelet collagen receptor glycoprotein (GP) VI or CLEC-2. The mechanisms underlying PLC regulation are not fully understood. An involvement of small GTPases of the Rho family (Rho, Rac, Cdc42) in PLC activation has been proposed but this has not been investigated in platelets. We here show that murine platelets lacking Rac1 display severely impaired GPVI- or CLEC-2-dependent activation and aggregation. This defect was associated with impaired production of inositol 1,4,5-trisphosphate (IP₃) and intracellular calcium mobilization suggesting inappropriate activation of PLCγ2 despite normal tyrosine phosphorylation of the enzyme. Rac1 ^−/− platelets displayed defective thrombus formation on collagen under flow conditions which could be fully restored by co-infusion of ADP and the TxA₂ analog U46619, indicating that impaired GPVI-, but not G-protein signaling, was responsible for the observed defect. In line with this, Rac1 ^−/− mice were protected in two collagen-dependent arterial thrombosis models. Together, these results demonstrate that Rac1 is essential for ITAM-dependent PLCγ2 activation in platelets and that this is critical for thrombus formation in vivo. Irina Pleines, Margitta Elvers, Amrei Strehl: these authors contributed equally to this work.     

Functional analysis of α1β1 integrin in human natural killer cells

Upon activation with interleukin (IL)-2 human natural killer (NK) cells acquire on their surface the α1β1 and α2β1 integrins and down-regulate the expression of α6β1. By employing α1β1-specific monoclonal antibody (mAb) HP-2B6, characterized in our laboratory, we examined the functional role of the α1β1 integrin in NK cells. Treatment with HP-2B6 mAb partially interfered with attachment of cultured NK cells to type I collagen, and combined with an anti-α2β1 (TEA 1/41) mAb, it completely abrogated cell adhesion to this extracelular matrix protein. In contrast, NK cell attachment to laminin was completely blocked by the anti-β1 LIA 1/2 mAb, but was unaffected by α1 and α2-specific mAb; as α3β1 and α6β1 were undetectable, the data indicate that the α1β1 integrin binding sites for type I collagen and laminin are different. Incubation with anti-α1 HP-2B6 or its F(ab')₂ fragments specifically induced a rapid homotypic aggregation of NK cells that was dependent on active metabolism, an intact cytoskeleton and the presence of divalent cations (Ca²⁺ and Mg²⁺); homotypic cell adhesion was selectively blocked by anti-CD18, CD11a or CD54 mAb. In addition, stimulation of cultured NK cells with the anti-α1 HP-2B6 enhanced TNF-α production and induced tyrosine phosphorylation of a 110-kDa protein. Pretreatment with specific inhibitors of protein tyrosine kinase (PTK) activity (tyrphostin 25 and herbimycin A) completely abrogated the functional effects induced by the anti-α1 HP-2B6 mAb. Our data show that ligation of the α1β1 integrin positively modulates IL-2-activated NK cell function via a PTK-dependent pathway.     

Species variability in platelet aggregation response to different agonists

Conflicting data on platelet function in animal species are reported in the literature. In this study, the response of buffalo, horse, pig and sheep platelets to different agonists was assessed. Blood samples were collected from the jugular vein of six healthy subjects of each species and platelet-rich plasma was obtained by centrifugation. Platelet aggregation responses to increasing doses of adenosine 5'-diphosphate (ADP), arachidonic acid, collagen, platelet activating factor (PAF) and ristocetin were measured by a turbidimetric method. Horse platelets were the most responsive to ADP, collagen and PAF, whereas sheep platelets were the most responsive to ristocetin. The response to arachidonic acid varied least between species. PAF was the most effective agonist, inducing a maximum aggregation response at a concentration of 1 micro M for platelets of each species. Conversely, concentrations of ristocetin higher than 1mg/ml induced a maximum aggregation response only with sheep and horse platelets. The different responses of platelets from the four animal species to various agonists may reflect either (1). structural differences (including composition of the platelet membrane and presence of specific agonist receptors), or (2). activation of distinct signalling pathways by the agonist.     

Platelet size distribution measurements as indicators of shear stress-induced platelet aggregation

The mechanisms underlying shear stress-induced platelet aggregation (SIPA) were investigated by measuring changes in the platelet size distributions resulting from the exposure of human platelet-rich plasma (PRP) to well-defined shear stresses in a modified viscometer. Exposure of PRP to a shear stress of 100 dyne/cm² for 1 min at 37°C resulted in the loss of single platelets, an overall shift in the distribution to larger particle sizes, and the generation of platelet fragments. Treatment of PRP prior to shearing with a monoclonal antibody directed against platelet glycoprotein (GP) IIb-IIIa (integrin α_IIbβ₃) at a concentration that completely inhibited ADP-induced platelet aggregation also inhibited SIPA. Furthermore, incubation of PRP with a recombinant fragment of von Willebrand factor (vWF) that abolishes ristocetin-induced platelet agglutination significantly inhibited but did not eliminate SIPA. Pretreatment of PRP with the tetrapeptides RGDS or RGDV, which constitute the GP IIb-IIIa peptide recognition sequences on fibrinogen and vWF, almost completely blocked platelet aggregation at 100 dyne/cm², whereas the negative control peptide RGES had no discernible effect. Finally, incubation of PRP with a monoclonal antibody directed against the platelet vitronectin receptor (integrin α_vβ₃) did not affect SIPA. These results indicate that both GP IIb-IIIa and GP Ib, the latter through its interaction with vWF, are required for SIPA at 100 dyne/cm²; that the interaction of GP IIb-IIIa with its adhesive ligands under shear stress can be inhibited by RGD-containing peptides; and that the vitronectin receptor on platelets, which shares the same β₃ subunit as GP IIb-IIIa, plays no role in SIPA. On the basis of these results, the assessment of platelet size distributions provides a sensitive and quantitative measurement for the study of SIPA.     

Integrin alpha2beta1 (α2β1) promotes prostate cancer skeletal metastasis

Men who die of prostate cancer (PCa) do so because of systemic metastases, the most frequent of which are within the skeleton. Recent data suggest that the colonization of the skeleton is mediated in part by collagen type I, the most abundant protein within the bone. We have shown that enhanced collagen I binding through increased expression of integrin α₂β₁ stimulated in vitro invasion and promoted the growth of PCa cells within the bone. Accordingly, we sought to determine whether α₂β₁ integrin is a potential mediator of skeletal metastasis. To examine whether α₂β₁ integrin mediates PCa metastasis, α₂ integrin was over-expressed in low-tumorigenic LNCaP PCa cells or selectively knocked-down in highly metastatic LNCaP_col PCa cells. We document that the over-expression of α₂ cDNA stimulated whereas α₂ shRNA inhibited the ability of transduced cells to bind to or migrate towards collagen in vitro. Correspondingly, α₂ integrin knock-down reduced the tumor burden of intra-osseous tumors compared to control-transduced cells. To investigate the clinical significance of α₂β₁ expression in PCa, α₂β₁ protein was measured in prostatic tissues and in soft tissue and bone metastases. The data demonstrate that α₂β₁ protein was elevated in PCa skeletal metastases compared to either PCa primary lesions or soft tissue metastases suggesting that α₂β₁ contributes to the selective metastasis to the bone. Taken together, these data support that α₂β₁ integrin is needed for the efficient metastasis of PCa cells to the skeleton.     

Alpha2beta1 integrin is the major collagen‐binding integrin expressed on human Th17 cells

Growing evidence indicates that collagen‐binding integrins are important costimulatory molecules of effector T cells. In this study, we demonstrate that the major collagen‐binding integrin expressed by human Th17 cells is alpha2beta1 (α2β1) or VLA‐2, also known as the receptor for collagen I on T cells. Our results show that human naïve CD4⁺ T cells cultured under Th17 polarization conditions preferentially upregulate α2β1 integrin rather than α1β1 integrin, which is the receptor for collagen IV on T cells. Double staining analysis for integrin receptors and intracellular IL‐17 showed that α2 integrin but not α1 integrin is associated with Th17 cells. Cell adhesion experiments demonstrated that Th17 cells attach to collagen I and collagen II using α2β1 integrin but did not attach to collagen IV. Functional studies revealed that collagens I and II but not collagen IV costimulate the production of IL‐17A, IL‐17F and IFN‐γ by human Th17 cells activated with anti‐CD3. These results identify α2β1 integrin as the major collagen receptor expressed on human Th17 cells and suggest that it can be an important costimulatory molecule of Th17 cell responses.     

Influence of catheter intervention on platelet function and activation in a pig model for endovascular embolization of arteriovenous malformations

Catheter interventions are associated with the risk of thromboembolism; however, the extent of platelet activation is not known. Samples from an arteriovenous malformation model (n?=?21 pigs) were examined. The pigs received a continuous infusion of 66?IU?kg^?1?h^?1 (n?=?11) or 100?IU?kg^?1?h^?1 (n?=?10) heparin applied 20?min after an initial bolus of 100?IU/kg. Platelet aggregation according to Born and ADVIA 120? platelet activation indices were used to study platelet function and activation. Samples were taken previous to vascular puncture, following vascular puncture, 20?min after application of heparin bolus, following placement of microcatheters and after endovascular embolization. Reactivity of platelets was increased after puncture of the vessels (ADP: P?<?0. 0001, collagen: P?=?0.0053). Further on activity of platelets was constrained by heparinization (ADP: P?<?0.0001, collagen P?<?0.0001). It can be concluded that the puncture of vessels yields the highest risk of thromboembolic complications.     

Type 2B and pseudo type 2B Von Willebrand disease; a report of three cases

Increased affinity of Von Willebrand factor (VWF) for its platelet receptor GPIb-GPIX complex is responsible of an hemorrhagic disease, which is the Von Willebrand disease (VWD) type 2B when the molecular abnormality is located on the VWF, and the platelet-type 2B VWD when the mutation concern the platelet receptor. Haemostatic abnormalities in these bleeding disorders are similar; prolonged bleeding time, fluctuating thrombocytopenia, decreased factor VIII-VWF complex, and an increased response to low dose of ristocetin in platelets rich plasma. High molecular weight VWF multimers are decreased. We report here 2 cases of type 2B VWD and 1 case of platelet type 2B VWD. The distinction between these 2 diseases was established by studying platelet aggregation with weak doses of ristocetin in mixtures of washed platelets (of normal control or patient)+poor platelets plasma (normal or patient). In one case, VWD 2B was discovered late in a 49 years old man, and the factor VIIIC-VWF complex was not diminished. The distinction between these two congenital diseases is important for the treatment of bleeding manifestations which need VWF concentrates infusions in type 2B VWD and administration of platelets concentrates in pseudo type 2B VWD.     

Platelet aggregation and platelet function analyzer 100 (PFA-100) closure time in camels—a comparative study with humans

Despite the very active coagulation system in camels, there are no previous studies on camel platelet functions. It is our aim to study camel platelet function using aggregometry, Platelet Function Analyzer (PFA100), and flow cytometry. A total of 103 camels, 19 males and 84 females, were studied. Their ages ranged from 5 to 20 years (mean±SD: 6.4±4.4 years). The results obtained were compared with healthy humans. Platelet aggregometry was undertaken in platelet-rich plasma in response to adenosine diphosphate (ADP), adrenaline, collagen, arachidonic acid, and ristocetin. Camel platelet function in whole blood was also tested using the PFA-100 and by flow cytometry using three human monoclonal antibodies (CD42, CD61, and CD62). Camel platelets failed to respond to arachidonic acid, adrenaline, and ristocetin. However, responses to ADP and collagen were obtained but were less than the human values. The addition of human plasma caused some enhancement of the aggregation responses to adrenaline and collagen but not ristocetin or arachidonic acid. However, the presence of human serum or heparin resulted in a very marked enhancement of the camel platelet aggregation responses to all agonists, except arachidonic acid. PFA-100 closure times of the collagen–ADP and the collagen–epinephrine cartridges were markedly longer than in humans. In the flow cytometry studies, camel platelets failed to respond to any of the human monoclonal antibodies with or without activation by ADP, thrombin, human plasma, or serum. This first study on camel platelet functions uncovered the distinction between camel and human platelet functions. The lack of platelet responses to certain aggregating agonists, their enhancement with human plasma and serum, as well as the prolongation of the PFA-100 closure times, add other unique characteristics to the biology of this interesting creature.     

Recognition of E-cadherin on epithelial cells by the mucosal T cell integrin αM290β7 (αEβ7)

The integrin α_M290β7 on the surface of a T cell hybridoma, MTC-1, mediated adhesion of these cells to the mouse epithelial cell line CMT93. This interaction was critically dependent on the presence of divalent cations; Mn²⁺ strongly promoted adhesion, Ca²⁺ was ineffective and Mg²⁺ gave intermediate results. Antibodies to molecules on the surface of CMT93 cells were tested for inhibition of adhesion. One monoclonal antibody (mAb) against E-cadherin, ECCD-2, was found to have significant inhibitory activity. Other mAb to E-cadherin and antibodies to other molecules had no effect. To show that inhibition by ECCD-2 was specific for adhesion mediated by α_M290β7, MTC-1 cells were induced to adhere to CMT93 via the LFA-1/ICAM-1 pathway. For this purpose, the epithelial cells were treated with interferon-γ and tumor necrosis factor-α to induce ICAM-1 expression and, in addition, α_M290β7 on MTC-1 cells was down-regulated by culturing the cells in the absence of transforming growth factor β. Under these circumstances adhesion of MTC-1 cells to CMT93 was inhibited by an antibody to LFA-1 but not by ECCD-2. Transfection of mouse L cells with cDNA for mouse E-cadherin enabled MTC-1 cells to adhere to them through the α_M290β7 integrin; this interaction was inhibited both by ECCD-2 and by blocking antibody against the integrin. These data strongly suggest that E-cadherin is a principal ligand for α_M290β7.     

Effects of Fullerene C60 Nanocomposites on Human Platelet Aggregation

The effects of fullerene C₆₀ nanocomposites on human platelet aggregation induced by ADP, ristocetin, and collagen were studied. The nanocomposite containing fullerene C₆₀ in polyvinyl pyrrolidone solution did not change platelet aggregation, while fullerene C₆₀ in crown ether and Twin-80 solutions inhibited ADP-induced platelet aggregation by 20 and 30%, respectively.     

VCAM-1 is internalized by a clathrin-related pathway in human endothelial cells but its α 4β 1 integrin counter-receptor remains associated with the plasma membrane in human T lymphocytes

Lymphocyte extravasation involves a step(s) of de-adhesion to allow trans- and subendothelial migration in response to inflammatory signals. We show here that ligated VCAM-1 was rapidly internalized (t_1/2 14.5 min) in ECV 304 endothelial cells and in TNF-α-primed human umbilical vein-derived endothelial cells (t_1/2 11.2 min). The process required energy (ATP), intracellular Ca²⁺ , an intact cytoskeletal network and active protein kinases. The internalization of VCAM-1 involved a clathrin-dependent pathway based on the observations that 1) it was inhibited in cells treated with lysosomotropic agents or with a hypertonic concentration of sucrose, and 2) internalized VCAM-1 colocalized with clathrin. In contrast, the cross-linked α₄β₁ integrin counter-receptor of VCAM-1 remained associated with the plasma membrane of purified peripheral T and Jurkat cells. Our results suggest a model where VCAM-1 would initially participate in the retention of T cells to the endothelium by binding α₄β₁ integrin. Lymphocyte de-adhesion would be facilitated as a result of the internalization of VCAM-1. The persistent cell surface expression of α₄β₁ integrin would allow the migrating T cells to interact with and receive signal(s) from its fibronectin ligand of the extracellular matrix.     

Inhibition of 5-hydroxytryptamine-induced and-amplified human platelet aggregation by ketanserin (R 41 468), a selective 5-HT2-receptor antagonist

Ketanserin, a selective 5-HT₂-receptor antagonist, inhibits the reversible aggregation induced by 5-hydroxytryptamine (5-HT) in human platelet-rich plasma (PRP). In this respect, the compound is equipotent to cyproheptadine and more active than methysergide (IC₅₀: 1.66×10^–8 M, 1.44×10^–8 M and 5.62×10^–8 M respectively). Ketanserin is active against 5-HT-induced platelet aggregation after bothin vitro and oral administration to human volunteers. At concentrations up to 500 times in excess of the IC₅₀ for 5-HT-induced platelet reactions, ketanserin does not affect the aggregation induced by ADP, epinephrine, collagen or Thrombofax^®, the prostaglandin biosynthesis of thrombin-stimulated platelets, nor the active uptake of¹⁴C-5-HT by platelets.5-Hydroxytryptamine amplifies the human platelet aggregation induced by threshold concentrations of ADP, collagen, epinephrine, norepinephrine and induced irreversible aggregation of platelets pre-sensitized with Thrombofax^®. This amplification by 5-hydroxytryptamine results in a platelet response typical for the potentiated agonist; for the combination of the monoamine with collagen, the serotonergic amplification results in enhanced aggregation, release of -TG and PF₄ and excessive formation of TXB₂. Ketanserin, after bothin vitro and oral administration to man reduces the amplified response to the level of the potentiated agonist.The present evidence suggests the presence of functional 5-HT₂ receptors on the human platelet, different from those involved in the uptake of the monoamine.     

Influence of storage temperature on the responsiveness of human platelets to agonists

To investigate the effects of storage temperature on the responsiveness to agonists of human platelets prepared from stored blood, we measured the aggregability and acid-base status of platelets from 96 healthy subjects before and after storage of whole blood at 4 degrees C and room temperature (RT) up to 48 hr. After 24 hr storage at 4 degrees C, there were no significant differences in agonist-induced platelet aggregability, compared to fresh specimens. When blood was kept at RT for 24 hr, all of the platelet samples showed non-responsiveness (< 20% aggregability) to epinephrine and 70% (67/96) revealed impaired responsiveness (20 to 60% aggregability) to adenosine diphosphate (ADP); there were no samples that showed impaired- or non-responsiveness to collagen or ristocetin. Among the 67 samples that showed impaired responsiveness to ADP after RT storage, 62 (93%) exhibited the loss of a secondary wave of aggregation in response to ADP. After storage of blood at RT for 48 hr (pH 6.81 +/- 0.06), mean values of maximal platelet aggregability to epinephrine, ADP, collagen, and ristocetin were 8%, 16%, 19%, and 70%, which were significantly lower than the corresponding mean values after storage of blood at 4 degrees C for 48 hr (pH 7.04 +/- 0.04) (ie, 66%, 69%, 102%, and 91%, p < 0.01). In summary, refrigerated storage of human blood improves the stability of platelet responsiveness to agonists. Storage at RT causes platelet non-responsiveness to epinephrine and disturbs the release reaction of endogenous ADP.     

Inhibition of 5-hydroxytryptamine-induced and-amplified human platelet aggregation by ketanserin (R 41 468), a selective 5-HT2-receptor antagonist

Ketanserin, a selective 5-HT₂-receptor antagonist, inhibits the reversible aggregation induced by 5-hydroxytryptamine (5-HT) in human platelet-rich plasma (PRP). In this respect, the compound is equipotent to cyproheptadine and more active than methysergide (IC₅₀: 1.66×10^–8 M, 1.44×10^–8 M and 5.62×10^–8 M respectively). Ketanserin is active against 5-HT-induced platelet aggregation after bothin vitro and oral administration to human volunteers. At concentrations up to 500 times in excess of the IC₅₀ for 5-HT-induced platelet reactions, ketanserin does not affect the aggregation induced by ADP, epinephrine, collagen or Thrombofax^®, the prostaglandin biosynthesis of thrombin-stimulated platelets, nor the active uptake of¹⁴C-5-HT by platelets.5-Hydroxytryptamine amplifies the human platelet aggregation induced by threshold concentrations of ADP, collagen, epinephrine, norepinephrine and induced irreversible aggregation of platelets pre-sensitized with Thrombofax^®. This amplification by 5-hydroxytryptamine results in a platelet response typical for the potentiated agonist; for the combination of the monoamine with collagen, the serotonergic amplification results in enhanced aggregation, release of -TG and PF₄ and excessive formation of TXB₂. Ketanserin, after bothin vitro and oral administration to man reduces the amplified response to the level of the potentiated agonist.The present evidence suggests the presence of functional 5-HT₂ receptors on the human platelet, different from those involved in the uptake of the monoamine.     

Schizonepeta tenuifolia inhibits collagen stimulated platelet function via suppressing MAPK and Akt signaling

The prevalence of cardiovascular diseases (CVDs) is increasing at a rapid pace in developed countries, and CVDs are the leading cause of morbidity and mortality. Natural products and ethnomedicine have been shown to reduce the risk of CVDs. Schizonepeta (S.) tenuifolia is a medicinal plant widely used in China, Korea, and Japan and is known to exhibit anti-inflammatory, antioxidant, and immunomodulatory activities. We hypothesized that given herbal plant exhibit pharmacological activities against CVDs, we specifically explored its effects on platelet function. Platelet aggregation was evaluated using standard light transmission aggregometry. Intracellular calcium mobilization was assessed using Fura-2/AM, and granule secretion (ATP release) was measured in a luminometer. Fibrinogen binding to integrin α_Ⅱbβ₃, was assessed using flow cytometry. Phosphorylation of mitogen-activated protein kinase (MAPK) signaling molecules and activation of the protein kinase B (Akt) was assessed using Western blot assays. S. tenuifolia, extract potently and significantly inhibited platelet aggregation, calcium mobilization, granule secretion, and fibrinogen binding to integrin α_Ⅱbβ₃. Moreover, all extracts significantly inhibited MAPK and Akt phosphorylation. S. tenuifolia extract inhibited platelet aggregation and granule secretion, and attenuated collagen mediated GPVI downstream signaling, indicating the potential therapeutic effects of these plant extracts on the cardiovascular system and platelet function. We suggest that S. tenuifolia extract may be a potent candidate to treat platelet-related CVDs and to be used as an antiplatelet and antithrombotic agent.     

Mechanism of hypocoagulative effect of low-intensity laser radiation

Aggregation of platelets and their role in the hypocoagulation syndrome was studied afterin vitro irradiation of blood with a laser. Thromboelastography was performed in platelet-rich and platelet-free plasma. Low-intensity laser radiation affected the coagulation system via platelets. It decreased platelet aggregation induced by ADP, collagen, epinephrine, ristocetin, platelet activating factor, and fibrinogen. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 126, No. 7, pp. 36–38, July, 1998