Ristocetin induces platelet aggregation: a morphological demonstration

Changes in the morphology of human platelets induced by ristocetin in platelet-rich plasma (PRP) have been analysed at the ultrastructural level by means of a tannic acid procedure. Studies were also undertaken to measure the release of serotonin. Modifications of the aggregation tests induced by apyrase, a monoclonal antibody (Mab) to GPIIb/IIIa and by EDTA were also investigated. Transmission electron microscopy revealed that ristocetin precipitated adhesive proteins on the platelet membrane. An electron-dense deposit was seen within 20 s after ristocetin was added. When experiments were carried out in the aggregometer cuvette during stirring, groups of platelets became activated, changed their shape, and finally aggregated releasing part of their contents. The morphology of aggregates did not differ from those formed in the presence of ADP. Aggregation studies demonstrated that a Mab to GPIIb/IIIa modified the extent and the rate of the aggregation curve when RIPA was performed in citrated platelet-rich plasma (c-PRP), while apyrase modifies the extent, but not the slope, of the curve. Neither the antibody nor apyrase modified RIPA when it was performed in PRP obtained in the presence of EDTA. All this evidence suggests that RIPA in c-PRP, besides reflecting the interaction of GPIb with vWF, may also test other mechanisms of the platelet function including: assembly of GPIIb/IIIa complex, interaction of fibrinogen with this glycoprotein complex, and possibly the release reaction.     

Von Willebrand factor contaminating porcine factor VIII concentrate (Hyate: C) causes platelet aggregation

Severe thrombocytopenia has been reported to occur in some patients treated with a polyelectrolyte-fractionated porcine factor VIII concentrate (Hyate:C) (Kernoff et al, 1984; Gatti & Mannucci, 1984). We report here that Hyate:C induces aggregation of platelet-rich plasma in vitro accompanied by ATP release and thromboxane B2 formation, and is inhibited by EDTA and prostaglandin E1. Hyate:C induced platelet aggregation is affected by monoclonal antibodies anti-glycoprotein Ib or anti-glycoprotein IIb/IIIa and suppressed by polyclonal anti-porcine von Willebrand factor antiserum. We conclude that the aggregating ability of Hyate:C is due to porcine von Willebrand factor present in the concentrate. The occasional thrombocytopenia described after Hyate:C infusions probably results from the interaction between platelets and the porcine von Willebrand factor contaminating the concentrate.     

First evidence: rivaroxaban and apixaban reduce thrombin-dependent platelet aggregation

Rivaroxaban and apixaban are direct oral anticoagulants whose target specificity is to activate factor X (FXa). It is still not fully understood how xabans impact platelet function. This single-center observational study aimed to assess in vitro platelet function in patients with atrial fibrillation receiving rivaroxaban or apixaban. It examined quantification of platelet aggregation assessed by light transmission aggregometry in thirty-four patients treated with apixaban or rivaroxaban. The thrombin-induced platelet aggregation was significantly lower 2 h after taking selected xabans compared to baseline value (69.55?±?32.15% vs. 44.79?±?34.97.9%; p?<?0.0001). This effect was only observed in patients who received rivaroxaban or apixaban for more than 1 week. The thrombin-induced platelet aggregation is reduced in cardiovascular patients receiving rivaroxaban or apixaban. This reduction is likely to depend on the duration of the treatment. Duration of treatment should be considered in future studies focusing on DOACs and platelet aggregation.     

Inhibition of human platelet aggregation and thromboxane-B2 production by melatonin: evidence for a diurnal variation

The effects of melatonin on platelet aggregation and thromboxane-B2 (TxB2) production induced by 1-4 x 10(-6) M adenosine diphosphate (ADP) or 0.6 x 10(-3) M arachidonic acid (AA) were assessed in platelet-rich plasma (PRP). Micromolar concentrations of melatonin inhibited in a dose-dependent way ADP-induced platelet aggregation with individual inhibitions 40% or more at 10(-6)-10(-5) M. A significant depression of AA-induced platelet aggregation was observed only at 10(-5)-10(-4) M melatonin. Morning (0830 h)-evening (1800 h) studies of ADP-induced platelet aggregation in seven normal men showed a higher sensitivity at 1800 h when analyzed as a global inhibitory effect of melatonin (P less than 0.01). Moreover, only during the evening hours did melatonin induce reversible aggregation, an index of inhibition of the platelet secretory process elicited by ADP exposure. No diurnal variability in melatonin inhibition of AA-induced aggregation was detected. TxB2 production elicited by AA in the evening was inhibited significantly in a concentration-related manner by a 2-min preincubation with 10(-9)-10(-5) M melatonin, while during the morning hours the inhibition was significant only at 10(-6) M or higher melatonin concentrations. In the case of ADP, the inhibition of TxB2 release attained significance at 10(-5)-M (0830 h) or 10(-6)-M concentrations (1800 h). In the presence of either stimulatory agent, melatonin depression of TxB2 generation was about 2-fold greater at 1800 h than at 0830 h. The diurnal changes in melatonin effect on TxB2 production were also observed in thrombin-stimulated washed platelets. The present data indicate the existence of circadian variations in platelet responsiveness to melatonin in humans.     

ADP induces partial platelet aggregation without shape change and potentiates collagen-induced aggregation in the absence of Galphaq

Platelets from Galphaq knockout mice are unable to aggregate in response to physiological agonists like adenosine 5'-diphosphate (ADP), thromboxane A(2), thrombin, or collagen, although shape change still occurs in response to all of these agonists except ADP. ADP-induced platelet aggregation results from simultaneous activation of the purinergic P2Y(1) receptor coupled to calcium mobilization and shape change and of a distinct P2 receptor, P2cyc, coupled through Gi to adenylyl cyclase inhibition, which is responsible for completion and amplification of the response. P2cyc could be the molecular target of the antithrombotic drug clopidogrel and the adenosine triphosphate (ATP) analogs AR-C69931MX, AR-C67085, and AR-C66096. The aim of the present study was to determine whether externally added ADP could still act through the Gi pathway in Galphaq-deficient mouse platelets and thereby amplify the residual responses to agonists such as thrombin or collagen. It was found that (1) ADP and adrenaline still inhibited cyclic AMP accumulation in Galphaq-deficient platelets; (2) both agonists restored collagen- but not thrombin-induced aggregation in these platelets; (3) the effects of ADP were selectively inhibited in vitro by the ATP analog AR-C69931MX and ex vivo by clopidogrel and hence were apparently mediated by the P2cyc receptor; and (4) high concentrations of ADP (100 micromol/L) induced aggregation without shape change in Galphaq-deficient platelets through activation of P2cyc. Since adrenaline was not able to induce platelet aggregation even at high concentrations, we conclude that the effects of ADP mediated by P2cyc are not restricted to the inhibition of adenylyl cyclase through Gi(2).     

Endotoxin-induced changes in human platelet membranes: morphologic evidence

Interaction between human platelets and bacterial endotoxin was studied in vitro with transmission and scanning electron microscopy. Washed human platelets, whose aggregation was blocked with apyrase, were incubated in a plasma-free medium containing crude endotoxin that had previously been complexed with copper. Thirty minutes of incubation resulted in adherence of endotoxin particles to the platelet surface, breaks in the platelet plasma membrane with apparent attempts at repair, pseudoped formation, and centralization of platelet organelles. Copper appeared to potentiate these phenomena, since neither Cu2+ at low concentrations nor endotoxin alone altered the morphology of the platelet membrane. This platelet-endotoxin interaction may be an intermediary step in the detoxification and clearance of endotoxin from the plasma.     

Solubilization of human platelet alpha-adrenergic receptors: evidence that agonist occupancy of the receptor stabilizes receptor--effector interactions

The alpha-adrenergic receptors of human platelet membranes can be directly identified by both a radiolabeled agonist, [3H]epinephrine, and a radiolabeled antagonist, [3H]yohimbine. Digitonin solubilizes a binding component from the membrane that is indistinguishable from the alpha-receptor identified in the native platelet membrane as assessed by (i) order of potency of agonists and antagonists and (ii) affinity of the receptor for [3H]-yohimbine and competing antagonists. However, the solubilized receptor demonstrates a reduced affinity for agonists and a loss of the ability of guanine nucleotides to modulate receptor affinity for agonists. Prelabeling of human platelet membranes with [3H]-epinephrine results in a guanine nucleotide-sensitive agonist-receptor complex that sediments more rapidly in sucrose gradients than do unoccupied or antagonist-occupied receptors. Thus, agonist occupancy of the alpha-receptor prior to membrane solubilization may promote or stabilize receptor interaction with effector components in the membrane, one of which may be the GTP regulatory protein responsible for modulation of receptor affinity.     

Impairment of platelet aggregation in hemolytic uremic syndrome: evidence for platelet "exhaustion"

Thrombocytopenia, microangiopathic hemolytic anemia, and acute renal failure are the hallmarks of hemolytic-uremic syndrome (HUS). This report presents the results on platelet studies from 10 consecutive HUS patients in childhood. During their acute illness, they all displayed a characteristic pattern of impaired platelet function: no aggregating responses to epinephrine, some to ADP, and moderate to collagen. In addition, platelet contents of beta-thromboglobulin (beta TG) were markedly reduced. As these patients improved clinically, their platelet- aggregating responses also normalized despite their uremic state. Incubation of platelets with uremic plasma or guanidino-succinic acid, a uremic toxin, had minor effects on platelet-aggregating activity. Since low levels of platelet beta TG suggest that these platelets were in an exhausted state, in vitro experiments were performed to exhaust normal platelets by incubation at 37 degrees C. A proportional impairment of platelet-aggregating responses and decreasing levels of platelet beta TG were noted. Furthermore, the pattern of impairment was similar to that found in the platelet-aggregating activities of HUS patients. Thus, "exhaustion," in addition to azotemia and thrombocytopenia, are factors that contribute to the functional impairment of platelets in these patients. Further studies to reveal mechanisms that lead to platelet exhaustion in HUS are of fundamental importance in the understanding of this illness.     

Serglycin proteoglycan deletion induces defects in platelet aggregation and thrombus formation in mice

Serglycin (SG), the hematopoietic cell secretory granule proteoglycan, is crucial for storage of specific secretory proteins in mast cells, neutrophils, and cytotoxic T lymphocytes. We addressed the role of SG in platelets using SG-/- mice. Wild-type (WT) but not SG-/- platelets contained chondroitin sulfate proteoglycans. Electron microscopy revealed normal alpha-granule structure in SG-/- platelets. However, SG-/- platelets and megakaryocytes contained unusual scroll-like membranous inclusions, and SG-/- megakaryocytes showed extensive emperipolesis of neutrophils. SG-/- platelets had reduced ability to aggregate in response to low concentrations of collagen or PAR4 thrombin receptor agonist AYPGKF, and reduced fibrinogen binding after AYPGKF, but aggregated normally to ADP. 3H-serotonin and ATP secretion were greatly reduced in SG-/- platelets. The alpha-granule proteins platelet factor 4, beta-thromboglobulin, and platelet-derived growth factor were profoundly reduced in SG-/- platelets. Exposure of P-selectin and alphaIIb after thrombin treatment was similar in WT and SG-/- platelets. SG-/- mice exhibited reduced carotid artery thrombus formation after exposure to FeCl3. This study demonstrates that SG is crucial for platelet function and thrombus formation. We propose that SG-/- platelet function deficiencies are related to inadequate packaging and secretion of selected alpha-granule proteins and reduced secretion of dense granule contents critical for platelet activation.     

The in vitro effects of verbascoside on human platelet aggregation

Preliminary in vitro and animal studies have shown that verbascoside, a phenolic compound, may have several favourable biological activities, including an influence on endothelial function and on platelet aggregation. We sought to evaluate the effects of verbascoside, biotechnologically produced from plant cell cultures, on human platelet aggregation (PA). The blood from 40 aspirin-na?ve volunteers with at least one cardiovascular risk factor was preincubated in vitro with verbascoside (1 and 2?mg/dL) and aspirin (100?μM). The blood from 20 patients with a prior diagnosis of coronary heart disease who were chronically assuming aspirin was preincubated in vitro with verbascoside (1 and 2?mg/dL). PA is measured with a light transmission aggregometry and multiplate analyzer. As compared to reference, preincubation with verbascoside resulted in a significant inhibition of adenosine diphosphate (ADP) and arachidonic acid (AA)-induced PA (p?相似文献   

Effect of vitamin C on platelet adhesiveness and platelet aggregation in coronary artery disease patients

The effect of oral administration of vitamin C on platelet adhesive index (PAI), platelet aggregate ratio (PAg R) and serum ascorbic acid levels was studied. Feeding 75 g of butter to healthy males (group I, n = 10 cases), enhanced the tendency of platelet adhesiveness (PAd) and platelet aggregation (PAg) to a significant level at the end of 4 h. This was distinctly prevented when 1 g of vitamin C was added to the fatty meal. In coronary artery disease (CAD) patients (group II, n = 20 cases) 10 days of vitamin C administration at 1 g every 8 hours decreased the PAd (p less than 0.001) and PAg (p less than 0.05) significantly. There was also a significant (p less than 0.001) rise in the vitamin C levels. The study brings out a property of vitamin C which may be of considerable importance in prevention of chronic thromboatherosclerotic disease of the arteries.     

Interaction of subcomponent C1q and collagen with isolated platelet membranes in platelet aggregation

Human platelet membranes were isolated by the glycerol-lysis technique. When tested by a turbidimetric method in the presence of fibrinogen and calcium ions, the membrane vesicles exhibited patterns of reactivity to collagen. As with human platelet-rich plasmas, the aggregating effects of collagen were inhibited by preincubating the membrane suspensions with hemolytically active human C1q. Upon addition to platelet-rich plasma, the isolated membranes reduced collagen-provoked platelet aggregation. This inhibitory effect was partially suspended by prior incubation of the membranes with native C1q. The results show that the receptors for collagen and C1q are preserved during the membrane isolation procedure and support the competitive nature of inhibition by C1q of collagen-induced platelet aggregation.     

Hydrogen sulphide pathway contributes to the enhanced human platelet aggregation in hyperhomocysteinemia

Homocysteine is metabolized to methionine by the action of 5,10 methylenetetrahydrofolate reductase (MTHFR). Alternatively, by the transulfuration pathway, homocysteine is transformed to hydrogen sulphide (H₂S), through multiple steps involving cystathionine β-synthase and cystathionine γ-lyase. Here we have evaluated the involvement of H₂S in the thrombotic events associated with hyperhomocysteinemia. To this purpose we have used platelets harvested from healthy volunteers or patients newly diagnosed with hyperhomocysteinemia with a C677T polymorphism of the MTHFR gene (MTHFR++). NaHS (0.1–100 µM) or l-cysteine (0.1–100 µM) significantly increased platelet aggregation harvested from healthy volunteers induced by thrombin receptor activator peptide–6 amide (2 µM) in a concentration-dependent manner. This increase was significantly potentiated in platelets harvested from MTHFR++ carriers, and it was reversed by the inhibition of either cystathionine β-synthase or cystathionine γ-lyase. Similarly, in MTHFR++ carriers, the content of H₂S was significantly higher in either platelets or plasma compared with healthy volunteers. Interestingly, thromboxane A₂ production was markedly increased in response to both NaHS or l-cysteine in platelets of healthy volunteers. The inhibition of phospholipase A₂, cyclooxygenase, or blockade of the thromboxane receptor markedly reduced the effects of H₂S. Finally, phosphorylated–phospholipase A₂ expression was significantly higher in MTHFR++ carriers compared with healthy volunteers. In conclusion, the H₂S pathway is involved in the prothrombotic events occurring in hyperhomocysteinemic patients.Hyperhomocysteinemia (HHcy) is a risk factor for neurovascular and cardiovascular disease associated with endothelial dysfunction and accelerated atherosclerosis (1–3). Many clinical and epidemiological studies have demonstrated a positive correlation between homocysteine (Hcy) plasma levels and cardiovascular disorders (4, 5), leading to the general conclusion that Hcy is a prothrombotic factor (6–8). However, the mechanism(s) through which elevated circulating levels of Hcy promote vascular disease and thrombosis is still unclear (9). Hcy has two primary fates: conversion through a reaction catalyzed by 5,10 methylenetetrahydrofolate reductase (MTHFR) into l-methionine or conversion to l-cysteine (l-Cys) via a transulfuration pathway (10, 11). The transulfuration pathway relies upon cystathionine β-synthase (CBS) to transform Hcy in cysthathionine, which is converted by cystathionine γ-lyase (CSE) into l-Cys. Thereafter, both enzymes convert l-Cys to generate hydrogen sulphide (H₂S) (12, 13). H₂S has been recognized as the third member of the family of gaseous transmitters (14), and it is present in human blood at micromolar concentrations (10–100 μM) (15). It rapidly travels through cell membranes without using any specific receptor/transporter or intracellular signaling proteins. CBS and CSE are differentially expressed in cardiovascular as well as in several other body districts (13). The physiological functions of H₂S are mediated by a variety of molecular targets, including ion channels and signaling proteins (16–18). Alterations in H₂S metabolism contribute to an array of cardiovascular disorders such as hypertension, atherosclerosis, heart failure, and diabetes (19). Nevertheless, the influence of H₂S on platelet function and, in turn, on blood clotting has been poorly explored. We hypothesized that H₂S could be involved in the thrombotic events associated with HHcy. To address this issue, we used human platelets harvested either from healthy volunteers or from patients with a C677T polymorphism of the MTHFR gene (MTHFR++) that is linked to HHcy (20–22).     

Effects of tomato extract on human platelet aggregation in vitro

Among all fruits tested in vitro for their anti-platelet property, tomato had the highest activity followed by grapefruit, melon, and strawberry, whereas pear and apple had little or no activity. Tomato extract (20-50 w l of 100% juice) inhibited both ADP- and collagen-induced aggregation by up to 70% but could not inhibit arachidonic acid-induced platelet aggregation and concomitant thromboxane synthesis under similar experimental conditions. The anti-platelet components (MW <1000 Da) in tomatoes are water soluble, heat stable and are concentrated in the yellow fluid around the seeds. The active fractions were separated using gel filtration and HPLC. The aqueous fraction (110 000 2 g supernatant) of tomatoes containing anti-platelet activity was subjected to gel filtration column chromatography (Biogel P2 column). The activity was fractionated into two peaks, peak-3 and peak-4 (major peak). Subsequently, peak-4 was further purified by HPLC using a reversed-phase column. NMR and mass spectroscopy studies indicated that peak F2 (obtained from peak 4) contained adenosine and cytidine. Deamination of peak F2 with adenosine deaminase almost completely abolished its anti-platelet activity, confirming the presence of adenosine in this fraction. In comparison, deamination of peak-4 resulted in only partial loss of inhibitory activity while the activity of peak-3 remained unaffected. These results indicate that tomatoes contain anti-platelet compounds in addition to adenosine. Unlike aspirin, the tomato-derived compounds inhibit thrombin-induced platelet aggregation. All these data indicate that tomato contains very potent anti-platelet components, and consuming tomatoes might be beneficial both as a preventive and therapeutic regime for cardiovascular disease.     

Tissue factor in microvesicles shed from U87MG human glioblastoma cells induces coagulation, platelet aggregation, and thrombogenesis

Microvesicles (diameter ca 200 nm) from the cell-free supernatant of U87MG human glioblastoma cell caused platelet aggregation and coagulation in a manner identical with that previously shown for the intact cells. Both activities were inhibited by dansylarginine -N-(3- ethyl-1,5-pentanediyl) amide (DAPA), confirming the thrombin-dependent nature of both activities. The specific activities per microgram of protein were 2-10 times greater in the microvesicles than in the plasma membrane fraction, suggesting localization in specific membrane domains. Sucrose density centrifugation gave a single protein peak (density 1.14) with congruent procoagulant and platelet aggregating activities. Both activities required the extrinsic pathway, as shown by studies with factor-deficient plasmas, and both were inhibited by heating (60 min/100 degrees C), by reduction and alkylation, and by incubation of the microvesicles with rabbit anti-bovine brain tissue factor antibody. These observations were confirmed using microvesicles from the HL-60 human promyelocytic leukemia cells, which are known to contain tissue factor activity. The results suggest that both procoagulant and proaggregating activities are causally related through the presence of tissue factor in the microvesicles. Studies with the Baumgartner perfusion apparatus showed that U87MG microvesicles increased the size of adherent thrombi nearly tenfold and that these thrombi were associated with nucleated cells from the blood. The increase in adherent thrombi did not occur if perfusion was carried out in the presence of DAPA, confirming the role of thrombin in their formation.