We investigated the combined effect of wall shear rate and immobilized collagen on platelet activation in flowing nonanticoagulated human blood. By combining an ex vivo model of thrombogenesis with flow cytometry, we showed that activated platelets can be detected in the bloodstream passing growing thrombi at a wall shear rate characteristic of moderately stenosed arteries (2600 s-1). The activation of the circulating platelets was clearly correlated with thrombus growth. Different antibodies against platelet activation-dependent surface markers had distinct sensitivity to the thrombotic process. alpha-Granule release detected by surface expression of CD62P seemed to be the most sensitive marker, as judged by both mean fluorescence intensity and fraction of platelets activated. The conformational change in glycoprotein IIb-IIIa, as detected by PAC-1, also seemed to be a sensitive marker and preceded binding of fibrinogen to activated glycoprotein IIb-IIIa, as detected by anti-fibrinogen. Large thrombi also elicited lysosome exocytosis, detected by surface expression of CD63. Finally, we observed a small decrease of glycoprotein Ib-IX expression, as detected by anti-CD42a. Thus, our study provides further information on the dynamics of platelet activation in relation to thrombus growth at arterial shear conditions in flowing nonanticoagulated human blood.

BACKGROUND

Piperlongumine (PL) is a compound isolated from the piper longum plant. It possesses anti-cancer activities through blocking the transcription factor STAT3 and by inducing reactive oxygen species (ROS) in cancer, but not normal cells. It also inhibits platelet aggregation induced by collagen, but the underlying mechanism is not known.

OBJECTIVE

We conducted in vitro experiments to test the hypothesis that PL regulates a non-transcriptional activity of STAT3 to specifically reduce the reactivity of human platelets to collagen.

RESULTS

PL dose-dependently blocked collagen-induced platelet aggregation, calcium influx, CD62p expression and thrombus formation on collagen with a maximal inhibition at 100 μM. It reduced platelet microvesiculation induced by collagen. PL blocked the activation of JAK2 and STAT3 in collagen-stimulated platelets. This inhibitory effect was significantly reduced in platelets pretreated with a STAT3 inhibitor. Although PL induced ROS production in platelets; quenching ROS using excessive reducing agents: 20 μM GSH and 0.5 mM L-Cysteine, did not block the inhibitory effects. The NADPH oxidase inhibitor Apocynin also had no effect.

CONCLUSIONS

PL inhibited collagen-induced platelet reactivity by targeting the JAK2-STAT3 pathway. We also provide experimental evidence that PL and collagen induce different oxidants that have differential effects on platelets. Studying these differential effects may uncover new mechanisms of regulating platelet functions by oxidants in redox signals.

The recovery of ischemic myocardium blood perfusion is the main treatment option for acute myocardial infarction (AMI). However, this treatment option has multiple side effects that directly affect the quality of life of the patients. The activation of platelet function plays an important role in the occurrence, development and treatment of AMI. The aim of the present study was to analyze the effects of remote ischemic post-conditioning on platelet activation of AMI patients with primary PCI treatment and clinical prognosis. A total of 71 patients with AMI were treated with primary percutaneous coronary intervention (PCI). They were randomly divided into control group (n=34) and observation group (n=37). The patients in the observation group were treated with remote ischemic post-conditioning. Further, flow cytometer was used to detect the platelet alpha granule membrane glycoprotein (CD62P) and the percentages of activated IIb/IIIa (PAC-1). The maximum platelet aggregation rate induced by adenosine diphosphate (ADP) and arachidonic acid (AA) was measured by light transmittance aggrometer. The incidence of major adverse cardiac events (MACE) was compared between the two groups during the follow-up period of 6 months. The percentage of CD62P (24 h after PCI) in the observation group was significantly lower than control group (P<0.05). Further, the incidence of MACE in the observation group was also lower than that of the control group (P<0.05). Remote ischemic post-conditioning could reduce the incidence of MACE in patients with AMI after primary PCI treatment. Moreover, the above observation may be related to the improvement of platelet CD62P activation.

Endothelial dysfunction is closely associated with hypertension. Protection of vascular endothelial cell is the key to prevention and treatment of hypertension. Uncaria rhynchophylla total alkaloids and Semen Raphani soluble alkaloid, isolated from traditional Chinese medicine Uncaria rbyncbopbylla and Semen Raphani respectively, exhibit properties of anti-hypertension and protection of blood vessels. In the present study, we observed the protective effect of the combined use of Uncaria rhynchophylla total alkaloids and Semen Raphani soluble alkaloid to the vascular endothelial cell in N'-nitro-L-arginine-induced hypertensive rats and investigate the preliminary mechanism. Blood pressure was detected by non-invasive rats tail method to observe the anti-hypertension effect of drugs. Scanning electron microscopy was used to observe the integrity or shedding state of vascular endothelial cell. The amount of circulating endothelial cells and CD54 and CD62P expression on circulating endothelial cells were tested to evaluate the endothelium function. In this study, we found that the Uncaria rhynchophylla total alkaloids and Semen Raphani soluble alkaloid compatibility can effectively lower the blood pressure, improve the structural integrity of vascular endothelium, and significantly reduce the number of circulating endothelial cells. Furthermore, the mean fluorescence intensity of CD54 and CD62P expressed showed decrease after the intervention of Uncaria rhynchophylla total alkaloids and Semen Raphani soluble alkaloid compatibility. In conclusion, the combination of effective components of the Uncaria rhynchophylla total alkaloids and Semen Raphani soluble alkaloid demonstrated good antihypertension effect and vascular endothelium protective effect. The preliminary mechanism of the protective effect may attribute to relieve the overall low-grade inflammation.

Exocytosis following platelet activation leads to translocation of CD62P (P-selectin), CD63, and thrombospondin from cytoplasmic granules to the cell surface membrane, where these molecules, serving as activation markers, can be detected by flow cytometry. We report the detectability of these molecules preformed--prior to platelet activation--inside the cytoplasm of resting platelets. Two different methods are described, using either methanol or the Fix&Perm kit for cell membrane permeabilization. In addition, interleukin(IL-)1 alpha is shown to be present in platelet cytoplasm after methanol treatment but not after permeabilization using Fix&Perm. Whenever cell surface positivity for a specific marker coincides with intracellular presence, blocking of the surface membrane sites prior to membrane permeabilization is required in order to obtain fluorescence intensity attributable to cytoplasmic staining only. Our data demonstrate the feasibility of the methods described for the detection of intracellular platelet molecules.

Patients with bronchial asthma develop various types of asthmatic response to bronchial challenge with allergen, such as immediate/early asthmatic response (IAR), late asthmatic response (LAR) or delayed asthmatic response (DYAR), because of different immunologic mechanisms. The DYAR, occurring between 24 and 56 hours after the bronchial allergen challenge (p < 0.01), differs from IAR and LAR in clinical as well as immunologic features. This study investigates the expression of CD molecules (markers) on the surface of particular cell populations in the peripheral blood and their changes during the DYAR. In 17 patients developing the DYAR (p < 0.01), the bronchial challenge with allergen was repeated 2-6 weeks later. The repeated DYAR (p < 0.001) was combined with recording of CD molecule expression on various types of blood cells by means of flow cytometry up to 72 hours after the challenge. The results were expressed in percent of the mean relative fluorescence intensity. The DYAR was accompanied by (a) increased expression of CD11b, CD11b/18, CD16,CD32, CD35, CD62E, CD62L, CD64, and CD66b on neutrophils; CD203C on basophils; CD25 and CD62L on eosinophils; CD14, CD16, CD64, and CD86 on monocytes; CD3, CD4, CD8, CD11a, CD18, and CD69 on lymphocytes; CD16, CD56, CD57, and CD94 on natural killer (NK) cells; and CD31, CD41, CD61, CD62P, and CD63 on thrombocytes and (b) decreased expression of CD18 and CD62L on eosinophils, CD15 on neutrophils, and CD40 on lymphocytes. These results suggest involvement of cell-mediated hypersensitivity mechanism, on participation of Th1- lymphocytes, neutrophils, monocytes, NK cells, and thrombocytes in the DYAR.

Platelets play an important role in metastasis of circulating tumor cells (CTCs). It has been demonstrated that hydroxyethyl starch (HES) inhibits platelets function. However, the effect of HES on CTCs in patients with colorectal cancer remains unclear. We compared the effects of HES 200/0.5 and HES 130/0.4 on CTCs and platelets activation of colorectal patients in this study. Additionally, the effects of HES 200/0.5 or HES 130/0.4 on metastasis ability of colon cancer cell line that stimulated by activated platelets have been explored. In vivo, 90 patients undergoing colorectal cancer radical surgery received randomly 15 mL/kg of HES 200/0.5 (n = 45) or HES 130/0.4 (n = 45) infusion before surgery. Platelet glycoprotein IIb/IIIa (GPIIb/IIIa), CD62P and platelets aggregation rate (PAR) were evaluated pre-, intra- and postoperatively. Cytokeratin-20 (CK-20) mRNA was detected by reverse transcriptase polymerase chain reaction before and after surgery. In vitro, colon cancer SW480 cells were incubated with activated platelets in the presence or absence HES 200/0.5 or HES 130/0.4. The metastasis ability of SW480 cells was assessed by Transwell assay. The results showed that CK-20 mRNA positive rate in HES 200/0.5 group after surgery was decreased significantly as compared to group HES 130/0.4 (χ (2) = 6.164, P = 0.013). Simultaneously, a more pronounced inhibition of platelets activation was observed in group HES 200/0.5. A positive correlation between platelets activation marker and CK-20 mRNA positive rate was found. In vitro, HES 200/0.5, but not HES 130/0.4, decreased the invasion and migration ability of SW480 cells that induced by activated platelets. Besides, the expression of GPIIb/IIIa, CD62P and PAR was inhibited more strongly in group HES 200/0.5 than those in group HES 130/0.4. In summary, we found that HES 200/0.5 significantly decreased CTCs of patients undergoing colorectal cancer radical surgery as compared to HES 130/0.4, which might be associated with inhibiting platelets activation of HES 200/0.5. Furthermore, HES 200/0.5, but not HES 130/0.4, reduced the metastatic potential of colon cell line stimulated by activated platelets through depressing platelets activation. Modulation of platelets activity may be a novel strategy to minimize the risk of metastasis during surgery.

OBJECTIVE

To investigate the effect of hyperbaric oxygen (HBO) on platelet physiology.

METHODS

Human platelets were exposed to HBO (97.7% O(2), balance CO(2) at 2.2 ata) or control (CON; 5% CO(2), balance air at 1 ata) for 90 min, and analyzed for aggregation, protein release, ()NO production, and activation.

RESULTS

HBO induced 29.8+/-3.0% of platelets to aggregate compared with CON (5.5+/-0.9%). Proteins observed to be released in greater abundance from HBO- compared with CON-treated platelets included 14-3-3 zeta and alpha-2-macroglobulin. Release of ()NO by platelets was unaffected following exposure to HBO, as was platelet activation as measured by surface expression of PECAM-1, CD62P and the activated form of alpha(IIB)beta(IIIa).

CONCLUSIONS

Exposure to HBO induces both platelet aggregation and protein release. Further study will better define the precise mechanisms and effects of HBO on platelet activation.

N-Alkylated chitosan (NACS) may improve the haemostatic efficiency of chitosan (CS). To study its coagulation capability and function, a series of NACS with various carbon chain lengths and substitution degrees (SD) of alkyl groups were synthesized and characterized by FTIR, NMR, and elemental analysis. Haemolysis and toxicity assays revealed that NACS showed good biocompatibility. In vitro blood clotting tests indicated that NACS had better haemostatic activity than CS, of which N-octadecyl CS with 3.85% SD showed the best results. Blood plasma coagulation tests showed that NACS was not favourable for activating coagulation factors. Platelet adhesion, intracellular Ca2+, and CD62p measurements demonstrated that the coagulation properties of NACS were not related to platelet activation. Erythrocyte adhesion examination indicated that blood coagulation of NACS may be attributable to its effects on erythrocytes. This study suggests that NACS is an ideal candidate for clotting.

Platelets play a role in both the innate and adaptive immune systems. Methods for detecting activated platelets and leukocyte-platelet aggregates (LPAs) are useful for basic and applied research concerning the role of platelets in inflammation and immune disorders. The aim of the study was to develop flow cytometric assays for detection of platelets binding to monocytes and neutrophils and for activated platelets in canine whole blood and to investigate the effect of physiologic agonists. Citrate anticoagulated whole blood was incubated with monoclonal antibodies against CD14 and CD61 for detection of LPAs, and the effect of various agonists was investigated. For detection of activated platelets, whole blood was incubated with monoclonal antibodies against CD62P and against a receptor-induced binding site on fibrinogen (CAP1) with CD61 as a platelet identifier. Isotype controls were prepared in parallel. The individual physiologic agonists ADP, collagen and epinephrine increased LPAs, CD62P and CAP1 binding only modestly. However, combinations of agonists gave more substantial increases. A dose-response relationship was seen using alpha- and gamma-thrombin, and ADP as agonists. In conclusion, we have developed flow cytometry assays to measure LPAs and platelet activation in canine whole blood, and have explored the effect of various physiologic agonists at different concentrations.

OBJECTIVE

Platelets prepared after holding of whole blood overnight at 22 degrees C have a well-preserved metabolism. However, the possibility that such prolonged incubation with active granulocytes may increase platelet activation has not been fully tested.

METHODS

We investigated this possibility by flow cytometric analysis of membrane glycoproteins (GPs) Ib and IIb/IIIa and the activation markers CD62P and CD63 in platelet concentrates (PCs) prepared from whole blood that was held for either 6 h without cooling plates (n = 20) or for 24 h on cooling plates of 1,4-butanediol (n = 20). PCs were prepared by the platelet-rich plasma method and analyzed on the second storage day.

RESULTS

Platelet yield and aggregation response to ristocetin, collagen and epinephrine + ADP were similar in both types of PCs, as was the mean fluorescence intensity for GPs Ib and IIb/IIIa. PCs prepared by the overnight-hold method did not differ from those obtained 6 h after collection in the percentage of platelets expressing CD62P (12.3 +/- 6.2% vs. 14.1 +/- 4.0%; p>> 0.1) or CD63 (9.8 +/- 6.4% vs. 8.8 +/- 3.6%; p>> 0.1).

CONCLUSIONS

Prolonged holding of whole blood at 22 degrees C prior to component preparation does not increase the level of platelet activation.

Platelets of patients with sickle cell disease (SCD) show evidence of mild activation in the non-crisis steady state and greater activation during vaso-occlusive crises (VOC). Prasugrel, a potent inhibitor of ADP-mediated platelet activation and aggregation, may be useful in attenuating VOC. We compared platelet responses to ADP stimulation in patients with SCD and healthy subjects before and after treatment with prasugrel. In a phase 1 study, platelet biomarker levels were assessed in 12 adult patients with SCD and 13 healthy subjects before and after 12 ± 2 days of 5.0 or 7.5 mg/day prasugrel. The following were determined in whole blood samples stimulated with 20 µM ADP: (i) percentages of monocytes and neutrophils with adherent platelets (cell-platelet aggregates); (ii) the relative number (mass) of platelets associated with each monocyte and neutrophil as reported by CD61 mean fluorescence intensity (MFI) of the monocyte-platelet and neutrophil-platelet aggregates; (iii) the percentages of platelets positive for surface expression of CD40 ligand (CD40L), P-selectin (CD62p) and activated glycoprotein IIb-IIIa (GPIIb-IIIa); and (iv) the percentages of platelets and monocyte-platelet aggregates positive for surface tissue factor (TF) expression. At baseline, there were no significant differences between cohorts in the percentages of platelets expressing activation biomarkers. Following 12 days of prasugrel administration, the percentages of platelets expressing activation biomarkers following ADP stimulation were reduced in both cohorts, and there were no significant differences between groups. Both patients with SCD and healthy subjects had significant reductions in the monocyte-platelet and neutrophil-platelet aggregate MFI and the percentage of platelets expressing P-selectin and activated GPIIb-IIIa (all p < 0.05). Healthy subjects also had significant reductions in monocyte-platelet aggregate percentages (p = 0.004), neutrophil-platelet aggregate percentages (p = 0.011) and the percentage of CD40L-positive platelets (p = 0.044) that were not observed in patients with SCD. Prasugrel administration to SCD patients attenuates ex vivo ADP-stimulated platelet activation as measured by the percentage of platelets positive for P-selectin and GPIIb-IIIa, thus reducing the proportion of platelets that may participate in aggregates. Furthermore, prasugrel decreases ex vivo ADP-stimulated platelet aggregation with monocytes and neutrophils as measured by the monocyte-platelet and neutrophil-platelet aggregate MFI. This implies that in the presence of prasugrel, fewer platelets adhere to monocytes and neutrophils, which may result in reducing cell-platelet aggregate size. Therefore, reduced platelet reactivity and decreased size of leukocyte-platelet aggregates suggest additional mechanisms by which prasugrel may provide benefit to patients with SCD and support further investigation of possible therapeutic benefits of prasugrel in this population.

BACKGROUND

Pathogen reduction technologies (PRTs) increase the safety of the blood supply, but are also associated with cell damage. Our aim was to investigate the effect of Mirasol PRT on platelet (PLT) concentrates stored in plasma and whether the use of a PLT additive solution (PAS) is able to improve in vitro quality.

METHODS

Twenty-two buffy coats (BCs) were pooled and split into two equal parts. To one half, 2 units of plasma were added, and to the other, 2 units of SSP+ PAS were added. Each part was equally split in half again (to resemble pooling five BCs) and PLT concentrates were prepared. One plasma PLT concentrate was Mirasol treated, and the other served as control; similarly, one SSP+ PLT concentrate was Mirasol treated, and the other not. PLT concentrates were stored for 8 days (n = 12).

RESULTS

Mirasol PRT led to elevated lactate production in PLT concentrates in plasma, giving lower pH values throughout storage. The use of SSP+ mostly abrogated this effect, and Mirasol-treated PLT concentrates in SSP+ had only slightly higher lactate production rates and annexin A5 binding as control PLT concentrates in plasma. However, irrespective whether plasma or SSP+ was used, Mirasol PRT led to higher CD62P expression and lower hypotonic shock response (HSR) scores.

CONCLUSIONS

Mirasol treatment leads to higher PLT activation and lower HSR scores both when stored in plasma or SSP+. However, if Mirasol-treated PLTs are stored in SSP+, lactate metabolism and annexin A5 binding are lower, showing that PAS can partly mitigate the effect of PRT. The clinical relevance of this finding needs to be demonstrated.

Polysulfonated macromolecules are known to bind selectins, adhesion membrane proteins which are broadly implicated in inflammation. Commercially available branched polyethylenimine (PEI) was reacted with chlorosulfonic acid to generate sulfonated PEI with varying degrees of sulfonation. Remaining unreacted amine groups were then used for straightforward conjugation with pyropheophoribide-a, a near-infrared photosensitizer. Photosensitizer-labeled sulfonated PEI conjugates inhibited blood coagulation and were demonstrated to specifically bind to cells genetically programmed to overexpress L-selectin (CD62L) or P-selectin (CD62P). In vitro, following targeting, selectin-expressing cells could be destroyed via photodynamic therapy.

OBJECTIVE

Abnormal platelet counts have been noticed in acquired immune deficiency syndrome (AIDS) patients. However, the actual state of platelets in AIDS is unclear. We hypothesize that platelets are activated and platelet-derived microparticles increase in murine AIDS.

METHODS

To elucidate the ethanol effects on platelets in murine AIDS, we studied four groups: control, murine AIDS, ethanol, and ethanol plus murine AIDS. Platelet CD62p as a platelet activation marker and CD61(+) microparticles as platelet microparticles (PMPs) were measured by flow cytometry.

RESULTS

Platelets were significantly activated in mice with murine AIDS and chronic ethanol consumption. Increased platelet CD62p expression and increased PMPs were most pronounced in advanced stages of murine AIDS. Chronic ethanol consumption persistently enhanced platelet activation and PMP formation.

CONCLUSIONS

Elevated platelet CD62p and PMPs may represent a pro-thrombotic status that have important pathological consequences.

Infection induces platelet activation and consumption, which leads to thrombocytopenia, enhances microvascular thrombosis, impairs microcirculation and eventually triggers disseminated intravascular coagulation (DIC). It is well characterized that endotoxemia results in a pro-inflammatory and pro-coagulatory state, which favors platelet activation. However the early, direct effects of endotoxemia on platelets have not been investigated so far. Therefore we aimed to determine the early effects of the endotoxin lipopolysaccharide (LPS) on platelet function in vivo. In a human endotoxemia model, 15 healthy volunteers were stimulated with LPS (2 ng/kg). Blood was drawn before, 10, 30 and 60 min after LPS challenge and platelet activation analyzed by flow cytometry (GPIIb/IIIa activation, surface CD62P and CD40L, intraplatelet reactive oxygen formation and platelet-leukocyte aggregates) and ELISA (sCD40L, sCD62P and CXCL4). In parallel, blood samples and platelets were spiked with LPS (50 pg/ml) in vitro and monitored over 60 min for the same platelet activation markers. In vitro platelet stimulation with LPS activated platelets independent of the presence of leukocytes and enhanced their adhesion to endothelial cells. In contrast, in vivo no increase in GPIIb/IIIa activation or surface expression of CD62P was observed. However, endotoxemia resulted in a significant drop in platelet count and elevated the plasma CXCL4 levels already 10 min after the LPS challenge. These data indicate that LPS rapidly activates platelets, leading to α-granule release and endothelial adhesion. This might explain the drop in platelet count observed at the onset of endotoxemia.

This report describes the effect of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on platelet production and platelet function in humans. Subjects with advanced solid tumors received PEG-rHuMGDF daily for up to 10 days. There was no increase in circulating platelet count at doses of 0.03 or 0.1 microgram/kg/d by day 12 of study. At doses of 0.3 and 1.0 microgram/kg/d there was a threefold median increase (maximum 10-fold) in platelet count by day 16. The platelets produced in vivo in response to PEG-rHuMGDF showed unchanged aggregation and adenosine triphosphate (ATP)-release responses in in vitro assays. Tests included aggregation and release of ATP in response to adenosine diphosphate (ADP) (10, 5, 2.5, and 1.25 mumol/L), collagen (2 micrograms/mL), thrombin-receptor agonist peptide (TRAP, 10 mumol/L) and ristocetin (1.5 mg/mL). Administration of aspirin to an individual with platelet count of 1,771 x 10(3)/L resulted in the typical aspirin-induced ablation of the normal aggregation and ATP-release response to stimulation with arachidonic acid (0.5 mg/mL), collagen, and ADP (2.5 and 1.25 mumol/L). There was no change in the expression of the platelet-surface activation marker CD62P (P-selectin) nor induction of the fibrinogen binding site on glycoprotein IIb/IIIa as reported by the monoclonal antibody, D3GP3. An elevation of reticulated platelets was evident after 3 days of treatment with PEG-rHuMGDF and preceded the increase in circulating platelet count by 5 to 8 days; this reflected the production of new platelets in response to PEG-rHuMGDF. At later time points, the mean platelet volume (MPV) decreased in a manner inversely proportional to the platelet count. Levels of plasma glycocalicin, a measure of platelet turnover, rose 3 days after the initial increase in the peripheral platelet count. The level of plasma glycocalicin was proportional to the total platelet mass, suggesting that platelets generated in response to PEG-rHuMGDF were not more actively destroyed. Thus, the administration of PEG-rHuMGDF, to humans, increased the circulating platelet count and resulted in fully functional platelets, which showed no detectable increase in reactivity nor alteration in activation status.

Polyphenol-rich fruit and vegetable intake has been associated with reduction in platelet hyperactivity, a significant contributor to thrombus formation. This study was undertaken to investigate the possible role of hippuric acid, a predominant metabolite of plant cyclic polyols, phenolic acids and polyphenols, in reduction of platelet activation-related thrombogenesis. Fasting blood samples were collected from 13 healthy subjects to analyse the effect of varying concentrations of hippuric acid (100 μM, 200 μM, 500 μM, 1 mM and 2 mM) on activation-dependant platelet surface-marker expression. Procaspase activating compound-1 (PAC-1) and P-selectin/CD62P monoclonal antibodies were used to evaluate platelet activation-related conformational changes and α-granule release respectively using flow cytometry. Platelets were stimulated ex vivo via the P2Y1/P2Y12- adenosine diphosphate (ADP) pathway of platelet activation. Hippuric acid at a concentration of 1 mM and 2 mM significantly reduced P-selectin/CD62P expression (p = 0.03 and p < 0.001 respectively) induced by ADP. Hippuric acid at 2 mM concentration also inhibited PAC-1 activation-dependant antibody expression (p = 0.03). High ex vivo concentrations of hippuric acid can therefore significantly reduce P-selectin and PAC-1 expression thus reducing platelet activation and clotting potential. However, although up to 11 mM of hippuric acid can be excreted in the urine per day following consumption of fruit, hippuric acid is actively excreted with a recorded Cmax for hippuric acid in human plasma at 250-300 μM. This is lower than the blood concentration of 1-2 mM shown to be bioactive in this research. The contribution of hippuric acid to the protective effects of fruit and vegetable intake against vascular disorders by the pathways measured is therefore low but could be synergistic with lowered doses of antiplatelet drugs and help reduce risk of thrombosis in current antiplatelet drug sensitive populations.

This ex vivo study was performed to evaluate the anti-platelet and anti-thrombogenic potential of shikimic acid (SA), a plant phenolic metabolite. Fasting blood samples were collected from 22 sedentary participants to analyse the effect of varying concentrations of SA (0.1 mM, 0.2 mM, 0.5 mM, 1 mM and 2 mM) on platelet surface-marker expression, platelet aggregation and biomarkers of thrombogenesis. Monocyte-platelet aggregates (CD14/CD42b) and platelet endothelial cell adhesion molecule-1 (PECAM-1 or CD31), effective indicators of thrombus formation were evaluated. Procaspase-activating compound 1 (PAC-1) and P-selectin or CD62P were used to assess platelet activation-related thrombogenesis. Adenosine diphosphate (ADP) was used to stimulate the P2Y1/P2Y12 pathway of platelet activation to mimic the in vivo thrombogenic pathway. Platelet aggregation studies utilised both ADP and collagen as exogenous platelet agonists to target both P2Y1/P2Y12 and GPVI pathways of thrombus formation. It was observed with flow cytometry that SA produced a significant antiplatelet effect on PAC-1 (p = 0.03 at 2 mM) and CD62P (p = 0.017, p = 0.036 at 1 mM and 2 mM respectively) expression in addition to lowering monocyte-platelet aggregate formation (p = 0.013, p < 0.01 and p < 0.01 at 0.5 mM, 1 mM and 2 mM respectively). SA at 1 mM concentration reduced PECAM-1 expression (p = 0.035), signifying a reduction to endothelial leucocyte migration during thrombus growth. SA did not demonstrate a platelet aggregation inhibitory effect by targeting the GPVI collagen pathway but reduced ADP induced platelet aggregation at 2 mM concentration (p < 0.01 at 2 mM). The results suggest that SA, an active metabolite of polyphenol-rich food intake, could play an important role in reducing platelet activation, aggregation related thrombus formation and biomarkers of thrombogenesis in sedentary individuals.

BACKGROUND

Thrombin generation assay is a convenient and widely used method for analysis of the blood coagulation system status. Thrombin generation curve (TGC) is usually bell-shaped with a single peak, but there are exceptions. In particular, TGC in platelet-rich plasma (PRP) can sometimes have two peaks.

OBJECTIVE

We sought to understand the mechanism underlying the occurrence of two peaks in the PRP thrombin generation curve.

METHODS

Tissue factor-induced thrombin generation in PRP and platelet-poor plasma (PPP) was monitored using continuous measurement of the hydrolysis rate of the thrombin-specific fluorogenic substrate Z-Gly-Gly-Arg-AMC. Expression of phosphatidylserine (PS) and CD62P on the surface of activated platelets was measured by flow cytometry using corresponding fluorescently labeled markers.

RESULTS

The addition of the P(2)Y(12) receptor antagonist MeS-AMP (160 µM), 83 nM prostaglandin E(1) (PGE(1)), or 1.6% DMSO to PRP caused the appearance of two peaks in the TGC. The PS exposure after thrombin activation on washed platelets in a suspension supplemented with DMSO, PGE(1) or MeS-AMP was delayed, which could indicate mechanism of the second peak formation. Supplementation of PRP with 1.6% DMSO plus 830 nM PGE(1) mediated the disappearance of the second peak and decreased the amplitude of the first peak. Increasing the platelet concentration in the PRP promoted the consolidation of the two peaks into one.

CONCLUSIONS

Procoagulant tenase and prothrombinase complexes in PRP assemble on phospholipid surfaces containing PS of two types--plasma lipoproteins and the surface of activated platelets. Thrombin generation in the PRP can be two-peaked. The second peak appears in the presence of platelet antagonists as a result of delayed PS expression on platelets, which leads to delayed assembly of the membrane-dependent procoagulant complexes and a second wave of thrombin generation.

OBJECTIVE

To provide an earlier diagnosis and efficiently treat acute rejection episodes (ARE) after renal transplantation, we studied its relationship to platelet activation.

METHODS

The peripheral blood levels of platelet surface glycoprotein (CD61), platelet activation-dependent granule membrane protein (CD62p), lysosomal enzyme glycoprotein (CD63), macula densa granule membrane glycoprotein (CD42a), and fibrinogen receptor monoclonal antibody (PAC-1) among 203 patients with uremia in various stages before and after transplantation were assayed by flow cytometry. The patients with ARE were prospectively and randomly assigned to either a treatment group with an antiplatelet activation agent or a control group.

RESULTS

The incidence of ARE was remarkably increased among patients with greater expression levels of platelet activation markers in peripheral blood preoperatively. The values of platelet activation markers were significantly higher among patients with ARE compared with those showing either normal graft function or acute tubular necrosis. The greater the increase in CD63, the worse the ARE. The expression levels of platelet activation markers decreased remarkably among the group treated with a platelet activation inhibitor in addition to antirejection therapy: the rejection reversal time shortened and the dose of antihuman thymocyte globulin (ATG) was lower. The sensitivity of platelet activation markers was better than its specificity.

CONCLUSIONS

Our studies demonstrated an association between platelet activation and ARE after renal transplantation. Platelet activation before transplantation can predict the occurrence of ARE. Platelet activation inhibitor therapy after transplantation improved ARE reversal.

BACKGROUND

Chronic kidney disease (CKD) is an established predictor of recurrent ischemic events in patients with coronary artery disease (CAD). This association has been partially ascribed to high post-treatment platelet reactivity (HPPR) according to platelet function testing. However, the influencing factors of HPPR are assay-dependent, and the relevant data of elderly patients with stable CAD are absent.

METHODS

310 elderly patients (>80years of age) with stable CAD taking prolonged maintenance clopidogrel (75mg/day) were studied. Maximal platelet aggregation rate (MPA%) with light transmittance aggregometry and Platelet Reactive Units (PRU) with VerifyNow (VN) P2Y12 system were obtained. Markers of platelet activation, including PAC-1 and CD62P, were also determined.

RESULTS

Patients on different stages of CKD presented similar MPA% and expression of PAC-1 and CD62P. Although severe CKD patients were more likely to present HPPR identified by VNP2Y12 (odds ratio: 1.85, p=0.038), multiple logistic regression diminished this effect (adjusted odds ratio: 1.19, p=0.642), and revealed anemia as a possible predictor of HPPR (adjusted odds ratio: 5.92, p=0.001). However, in a parallel way, hemoglobin correlated with baseline PRU values as well as with post-treatment values (r=-0.624 and r=-0.463, respectively, p<0.001). Association between hemoglobin and PRU inhibition rate was not found. Moreover, hemoglobin exerted no influence on MPA% at all.

CONCLUSIONS

CKD is not necessarily associated with reduced antiplatelet effects of clopidogrel in elderly patients with stable CAD taking prolonged maintenance clopidogrel, and the seemingly influence of CKD on HPPR assessed by VNP2Y12 assay may be due to the artifactual effect of hemoglobin on VNP2Y12.

OBJECTIVE

To explore the effects of triple antiplatelet therapy on platelet aggregation and activation in patients who underwent coronary stenting.

METHODS

120 in-hospital coronary heart disease patients with coronary stenting were randomized to two groups receiving either triple antiplatelet drugs of aspirin and clopidogrel combined with cilostazol or dual antiplatelet drugs of aspirin and clopidogrel. On the first day after stenting cilostazol was added to the triple group patients who were previously administered aspirin and clopidogrel. Expressions of PAC-1 and CD62p which indicate platelet activation were assessed with flow cytometry and the maximal platelet aggregation rate (MPAR) induced by 5 and 20 micromol/L ADP was measured at the day before receiving cilostazol and the fifth day after stenting, respectively.

RESULTS

The baseline clinical characteristics did not differ significantly between the two groups. There were no significant differences in the baseline level of MPAR CD62p and PAC-1 at the first day after stenting between the two groups. The margins between the two measurements were [(6.44 +/- 14.44)% vs (5.41 +/- 13.77)%, P>> 0.05] for DeltaMPAR induced by 5 micromol/L ADP, [(8.50 +/- 15.50)% vs (7.84 +/- 14.21)%, P>> 0.05] for DeltaMPAR induced by 20 micromol/L ADP, [(5.12 +/- 11.25)% vs (1.08 +/- 4.97)%, P < 0.05] for DeltaCD(62)p and [(12.12 +/- 12.30)% vs (2.22 +/- 15.15)%, P < 0.01] for DeltaPAC-1 in the triple and dual group patients, respectively. Among the above measurements, DeltaCD62p and DeltaPAC-1 in the triple group patients were significantly higher than those in the dual group patients although DeltaMPAR did not significantly differ between the two groups at the fifth day after stenting. Subgroup analysis for patients with acute coronary syndrome (ACS) showed that DeltaMPAR induced by 5 micromol/L ADP [(8.68 +/- 10.35)% vs (2.92 +/- 13.06)%, P = 0.018], DeltaMPAR induced by 20 micromol/L [(11.05 +/- 11.14)% vs (5.16 +/- 13.27)%, P = 0.019], DeltaCD62p [(5.57 +/- 12.08)% vs (1.35 +/- 4.42)%, P = 0.028] and DeltaPAC-1 [(11.62 +/- 12.73)% vs (1.29 +/- 15.73)%, P = 0.001] in triple group were significantly higher than that in dual group. At 3-month clinical follow-up, the rate of major adverse cardiac and cerebral events was 0 in the triple group and 3.3% (2/60) in the dual group, and the rate of hemorrhage was 5% (3/60) in the triple group and 3.3% (2/60) in the dual group, the differences were not statistically significant.

CONCLUSIONS

Compared with dual antiplatelet regimen with aspirin plus clopidogrel, triple antiplatelet therapy with aspirin and clopidogrel combined with cilostazol is more efficient in inhibiting platelet activation and aggregation after coronary stent implantation. Large scale clinical trials are needed to confirm efficacy and safety of the triple antiplatelet regimen.

Using flow cytometry, platelet-leukocyte aggregate (PLA) formation has previously been documented in dogs with a variety of systemic inflammatory disorders and immune-mediated haemolytic anaemia. Platelet activation and subsequent interaction between platelets and leukocytes are important for regulating innate immunity and systemic inflammation. The objective of this study was to investigate PLA formation in canine babesiosis and to determine whether it was associated with outcome. Blood was collected from 36 client-owned dogs diagnosed with Babesia rossi infection and 15 healthy controls using EDTA as anticoagulant. Activated platelets and PLA formation were detected by measuring surface expression of P-selectin (CD62P) on platelets, monocytes and neutrophils. Of the Babesia-infected dogs, 29 survived and seven died. The percentage of CD62P-positive monocytes was significantly higher (P = 0.036) in the Babesia-infected dogs (54%) than in healthy control dogs (35.3%). However, there were no significant differences between the Babesia-infected and control groups for CD62P-positive platelets (4.9% and 1.2%, respectively) and CD62P-positive neutrophils (28.3% and 17.9%, respectively). The percentage of CD62P-positive monocytes was significantly higher (P = 0.019) in the survivors (58.9%) than in healthy control dogs; however, there were no significant differences between the non-survivors (39.2%) and the controls or between survivors and non-survivors. There were no significant differences between groups for the percentage of CD62P-positive platelets (survivors 4.8%; non-survivors 5.3%; controls 1.2%) or CD62P-positive neutrophils (survivors 31.6%; non-survivors 5.6%; controls 17.9%). In conclusion, Babesia-infected dogs, specifically dogs that survived, had a significantly increased percentage of platelet-monocyte aggregates compared to healthy control dogs.