Secondary Platelet Aggregation Research Articles

Ablation of CD38 Alleviates the Secondary Aggregation of Platelets

Ablation of CD38 Alleviates the Secondary Aggregation of Platelets

The tyrosine kinase receptor Tyro3 enhances lifespan and neuropeptide Y (Npy) neuron survival in the mouse anorexia (anx) mutation.

ABSTRACTSevere appetite and weight loss define the eating disorder anorexia nervosa, and can also accompany the progression of some neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS). Although acute loss of hypothalamic neurons that produce appetite-stimulating neuropeptide Y (Npy) and agouti-related peptide (Agrp) in adult mice or in mice homozygous for the anorexia (anx) mutation causes aphagia, our understanding of the factors that help maintain appetite regulatory circuitry is limited. Here we identify a mutation (C19T) that converts an arginine to a tryptophan (R7W) in the TYRO3 protein tyrosine kinase 3 (Tyro3) gene, which resides within the anx critical interval, as contributing to the severity of anx phenotypes. Our observation that, like Tyro3−/− mice, anx/anx mice exhibit abnormal secondary platelet aggregation suggested that the C19T Tyro3 variant might have functional consequences. Tyro3 is expressed in the hypothalamus and other brain regions affected by the anx mutation, and its mRNA localization appeared abnormal in anx/anx brains by postnatal day 19 (P19). The presence of wild-type Tyro3 transgenes, but not an R7W-Tyro3 transgene, doubled the weight and lifespans of anx/anx mice and near-normal numbers of hypothalamic Npy-expressing neurons were present in Tyro3-transgenic anx/anx mice at P19. Although no differences in R7W-Tyro3 signal sequence function or protein localization were discernible in vitro, distribution of R7W-Tyro3 protein differed from that of Tyro3 protein in the cerebellum of transgenic wild-type mice. Thus, R7W-Tyro3 protein localization deficits are only detectable in vivo. Further analyses revealed that the C19T Tyro3 mutation is present in a few other mouse strains, and hence is not the causative anx mutation, but rather an anx modifier. Our work shows that Tyro3 has prosurvival roles in the appetite regulatory circuitry and could also provide useful insights towards the development of interventions targeting detrimental weight loss.

Ex vivo recapitulation of trauma-induced coagulopathy and preliminary assessment of trauma patient platelet function under flow using microfluidic technology.

Relevant to trauma-induced coagulopathy diagnostics, microfluidic assays allow controlled hemodynamics for testing of platelet and coagulation function using whole blood. Hemodilution or hyperfibrinolysis was studied under flow with modified healthy whole blood. Furthermore, platelet function was also measured using whole blood from trauma patients admitted to a Level I trauma center. Platelet deposition was measured with PPACK-inhibited blood perfused over collagen surfaces at a wall shear rate of 200 s, whereas platelet/fibrin deposition was measured with corn trypsin inhibitor-treated blood perfused over tissue factor (TF)/collagen. In hemodilution studies, PPACK-treated blood displayed almost no platelet deposition when diluted to 10% hematocrit with saline, platelet-poor plasma, or platelet-rich plasma. Using similar dilutions, platelet/fibrin deposition was essentially absent for corn trypsin inhibitor-treated blood perfused over TF/collagen. To mimic hyperfibrinolysis during trauma, exogenous tissue plasminogen activator (50 nM) was added to blood before perfusion over TF/collagen. At both venous and arterial flows, the generation and subsequent lysis of fibrin were detectable within 6 minutes, with lysis blocked by addition of the plasmin inhibitor, ε-aminocaproic acid. Microfluidic assay of PPACK-inhibited whole blood from trauma patients revealed striking defects in collagen response and secondary platelet aggregation in 14 of 21 patients, whereas platelet hyperfunction was detected in three of 20 patients. Rapid microfluidic detection of (1) hemodilution-dependent impairment of clotting, (2) clot instability because of lysis, (3) blockade of fibrinolysis, or (4) platelet dysfunction during trauma may provide novel diagnostic opportunities to predict trauma-induced coagulopathy risk.

Detection of platelet sensitivity to inhibitors of COX-1, P2Y1, and P2Y12 using a whole blood microfluidic flow assay

Microfluidic devices recreate the hemodynamic conditions of thrombosis. Whole blood inhibited with PPACK was treated ex vivo with inhibitors and perfused over collagen for 300 s (wall shear rate=200 s(-1)) using a microfluidic flow assay. Platelet accumulation was measured in the presence of COX-1 inhibitor (aspirin, ASA), P2Y₁ inhibitor (MRS 2179), P2Y₁₂ inhibitor (2MeSAMP) or combined P2Y1 and P2Y₁₂ inhibitors. High dose ASA (500 μM), 2MeSAMP (100 μM), MRS 2179 (10 μM), or combined 2MeSAMP and MRS 2179 decreased total platelet accumulation by 27.5%, 75.6%, 77.7%, and 87.9% (p<0.01), respectively. ASA reduced secondary aggregation rate between 150 and 300 s without effect on primary deposition rate on collagen from 60 to 150 s. In contrast, 2MeSAMP and MRS 2179 acted earlier and reduced primary deposition to collagen between 60 and 105 s and secondary aggregation between 105 and 300 s. R(COX) and R(P2Y) (defined as a ratio of secondary aggregation rate to primary deposition rate) demonstrated 9 of 10 subjects had R(COX)<1 or R(P2Y)<1 following ASA or 2MeSAMP addition, while 6 of 10 subjects had R(P2Y)<1 following MRS 2179 addition. Combined MRS 2179 and 2MeSAMP inhibited primary platelet deposition rate and platelet secondary aggregation beyond that of each individual inhibitor. Receiver-Operator Characteristic area under the curve (AUC) indicated the robustness of R(COX) and R(P2Y) to detect inhibition of secondary platelet aggregation by ASA, 2MeSAMP, and MRS 2179 (AUC of 0.874 0.966, and 0.889, respectively). Microfluidic devices can detect platelet sensitivity to antiplatelet agents. The R-value can serve as a self-normalized metric of platelet function for a single blood sample.

Impact of Anemia on Platelet Response to Clopidogrel in Patients Undergoing Percutaneous Coronary Stenting

High residual platelet reactivity (HRPR) on clopidogrel is a predictor of recurrent ischemic events in patients undergoing percutaneous coronary interventions (PCI). Significant intraindividual variability in platelet aggregation on repeat testing has been reported. To understand factors contributing to the variability in platelet aggregation testing, we examined clinical and laboratory elements linked to HRPR in 255 consecutive patients tested ≥12 hours after PCI using light transmission aggregometry (LTA) in response to adenosine diphosphate 5 μmol/L and VerifyNow P2Y12 assay (VNP2Y12; Accumetrics). HRPR was defined as >46% residual aggregation for LTA and >236 P2Y12 response units (PRUs) for VNP2Y12. On multivariate analysis the only variable independently associated with HRPR with both LTA and VNP2Y12 was laboratory-defined anemia. Prevalences of HRPR by LTA were 34.3% in anemic patients, 15.6% in patients with normal hemoglobin levels, and 59.8% versus 25.9% by VNP2Y12 (p <0.005 for the 2 comparisons). In a subgroup of 50 patients, testing was done before and after the clopidogrel loading dose. At baseline there were no differences in platelet aggregation with either assay; however, absolute decrease in reactivity after the clopidogrel load was significantly less in anemic patients compared to patients with normal hemoglobin (change in residual aggregation by LTA 15.8 ± 5.8% vs 28.8 ± 3.2%, p <0.05; change in PRU by VNP2Y12 56.5 ± 35.5 vs 145.0 ± 14.2 PRUs, p <0.05, respectively). In conclusion, anemia is an important contributor to apparent HRPR on clopidogrel and may explain some of the intraindividual variability of platelet aggregation testing.

The inhibitory effects of cannabinoids, the active constituents of Cannabis sativa L. on human and rabbit platelet aggregation.

Olivetol, cannabigerol (CBG), cannabidiol (CBD), cannabinol (CBN) and tetrahydrocannabinol (delta 1-THC) were assessed for their ability to inhibit agonist-induced platelet aggregation and [14C]5-HT release. With the exception of olivetol, (40% maximal effectiveness), none of the compounds inhibited tetradecanoylphorbolacetate (TPA)-induced aggregation of human or rabbit platelets. All of these cannabinoids partially inhibited primary aggregation and totally inhibited secondary aggregation of human platelets when adrenaline was used as the agonist. Inhibition was dose-dependent over the range 10(-3)-10(-5) M. Both rabbit and human platelet aggregation induced by adenosine diphosphate was inhibited in a dose-dependent manner and the order of potency was CBG greater than CBD greater than olivetol greater than THC greater than CBN, the IC50 of CBG being 2.7 x 10(-4) M. PAF-induced aggregation of rabbit platelets was also inhibited by these compounds in a dose-dependent manner over the concentration range 10(-3) to 10(-4) M, however [14C]5-HT release was only partially prevented by the cannabinoids in a manner which did not correlate with inhibition of aggregation.

Abstract 5301: Fractalkine Desensitizes Platelets to Platelet Inhibition by the Endogenous Platelet Inhibitor Prostacyclin

Background: Fractalkine plays a pivotal role in atherogenesis. Platelet activation is a central factor in atherosclerosis progression. We previously reported that fractalkine induces platelet degranulation and P-selectin expression facilitating platelet-leukocyte adhesion. Fractalkine attenuates the bioavailability of the important endogenous platelet inhibitor nitric oxide. Methods: The influence of preincubation of platelets with fractalkine (1 μ g/ml) on platelet inhibition by the platelet inhibitor prostaglandin E1 (PGE1) was assessed by two assays: first, the ability to prevent PGE1-induced attenuation of ADP-induced platelet aggregation, and second, the capability to decrease PGE1-induced vasodilator-stimulated phosphoprotein (VASP)-phosphorylation determined by flow-cytometry. Samples were preincubated with fractalkine and stimulated with different concentrations of PGE1. Results: PGE1 significantly inhibited ADP-induced platelet aggregation. In samples pre-treated with fractalkine, prevention of ADP-induced platelet aggregation by PGE1was abrogated. While secondary platelet aggregation was prevented by PGE1 (1 μ M), preincubation with fractalkine for 10 minutes reestablished secondary platelet aggregation. These results indicate that fractalkine desensitized platelets to PGE1. PGE1-induced VASP-phosphorylation was significantly attenuated by preincubation with fractalkine in a time- and concentration-dependent pattern, whereby a maximum effect was achieved after preincubation of platelets with fractalkine for 10 minutes, which reduced PGE1 (1 μ M)-induced VASP-phosphorylation by ~25% (p&lt;0.01 vs. PGE1 without fractalkine). Conclusion: Fractalkine desensitizes platelets to PGE1 suggesting that platelet inhibition by the endogenous platelet inhibitor prostacyclin is affected by fractalkine. These results indicate a new pathomechanism by which this atherogenic chemokine facilitates relevant steps leading to platelet activation and leukocyte recruitment, namely rendering platelets less responsive to endogenous platelet inhibition.

Gefitinib Affects Functions of Platelets and Blood Vesselsvia Changes in Prostanoids Balance

Prostaglandins (PGs) and thromboxane (TX) produced by cyclooxygenase (COX) have a great influence on vascular systems and platelet functions. The serum levels of epidermal growth factor (EGF) and PGs were measured in patients with lung cancer treated with gefitinib, and the influence of EGF on platelet aggregation was investigated. Twenty patients were investigated. The serum level of TXB(2) increased significantly in all patients who received gefitinib for 2 weeks (before vs. after = 94.1 +/- 47.3 vs. 190.9 +/- 54.3, p<0.01). TXB(2) also increased significantly in responders without concurrent chemotherapy (before vs. after = 79.3 +/- 35.5 vs. 194.5 +/- 58.1, p<0.05), but not in non-responders (before vs. after = 106. 5 +/- 65.8 vs. 162.2 +/- 52.8, N.S.). PG 6-keto F1alpha and PGE(2) did not exhibit significant changes. Furthermore, EGF showed no significant change (after vs. before = 234 +/- 35 vs. 276 +/- 72, N.S.). Although there was no correlation between the levels of EGF and TXB(2) (N.S.), the PG 6-keto F2alpha/TXB(2) ratio decreased significantly (before vs. after = 0.054 +/- 0.018 vs 0.033 +/- 0.015, p<0.05). The secondary platelet aggregation observed after high-dose adenosine diphosphate stimulation was inhibited after a 1-minute preincubation with EGF. Platelet aggregation in patients after gefitinib administration tended to accelerate and secondary aggregation was observed after low-dose adenosine diphosphate stimulation. We conclude that careful observation is needed for patients with chronic obstructive pulmonary disease, pulmonary fibrosis, and thromboembolic diseases receiving gefitinib. Furthermore, measurement of prostanoids may be a good predictor of the beneficial and adverse effects. Moreover, the combination of gefitinib with a COX inhibitor that regulates TXA(2)/PGI(2) balance should be evaluated.

Thiopental enhances human platelet aggregation by increasing arachidonic acid release

Conflicting results have been reported regarding the effect of thiopental on aggregation and cytosolic calcium levels in platelets. The present study attempted to clarify these phenomena. Using platelet-rich plasma or washed suspensions, platelet aggregation, thromboxane (TX) B2 formation, arachidonic acid (AA) release, and cytosolic free calcium concentrations ([Ca2+]i) were measured in the presence or absence of thiopental (30-300 microM). Platelet activation was induced by adenosine diphosphate (ADP, 0.5-15 microM), epinephrine (0.1-20 microM) arachidonic acid (0.5-1.5 mM), or (+)-9,11-epithia-11,12-methano-TXA2 (STA2, 30-500 nM). Measurements of primary aggregation were performed in the presence of indomethacin (10 microM). Low concentrations of ADP and epinephrine, which did not induce secondary aggregation in a control study, induced strong secondary aggregation in the presence of thiopental (> or = 100 microM). Thiopental (> or = 100 microM) also increased the TXB2 formation induced by ADP and epinephrine. Thiopental (300 microM) increased ADP- and epinephrine-induced 3H-AA release. Thiopental (300 microM) also augmented the ADP- and epinephrine-induced increases in [Ca2+]i in the presence of indomethacin. Thiopental appears to enhance ADP- and epinephrine-induced secondary platelet aggregation by increasing AA release during primary aggregation, possibly by the activation of phospholipase A2.

Disaggregation of aggregated platelets by apyrase from the tick, Ornithodoros savignyi (Acari: Argasidae).

Apyrase, secreted by ticks during feeding, is a platelet aggregation inhibitor that functions as a regulator of the host's hemostatic system. This present study concerns the disaggregation effect of salivary gland apyrase from the tick Ornithodoros savignyi. Secondarily aggregated platelets, disaggregated by apyrase, exhibited a reversal of shape from a spherical (aggregated) form to a discoid form, reminiscent of reversible aggregation at low ADP concentrations in citrated platelet-rich plasma. However, they showed a dilatory open canaliculary system and an absence of granules indicating disaggregation after degranulation had taken place. In contrast, disaggregation by the fibrin(ogen)olytic enzyme, plasmin, showed that platelets degranulated, but retained a spherical form with numerous extended pseudopods. While thrombin had no effect on aggregation or clotting of platelets disaggregated with plasmin, it did activate those platelets disaggregated with apyrase and clotted the plasma. This is the first study to describe the disaggregating effects of tick derived apyrase on aggregated platelets. It also shows that apyrase can disaggregate platelets even after secondary aggregation and degranulation of platelets has taken place. Platelet aggregation is one of the main barriers encountered by ticks during feeding and counteraction of this process by ticks is an important factor for successful feeding.

The effects of light to moderate drinking on cardiovascular diseases.

3 Department of Family Preventive Medicine, UCSD, La Jolla, CA, USA; 4 Brigham and Womens Hospital, Boston, MA, USA; 5 Danish Epidemiology Science Center, Institute of Preventive Medicine, Kommunehospitalet, Copenhagen, Denmark; 6 Department of Community Health, University of Aukland, NZ; 7 Kaiser Permanente Medical Center, Oakland, CA, USA; 8 University of Kuopio, Kuopio, Finland; and 9 Royal Free and University College Medical School, London, UK

Case studies in therapeutics: warfarin resistance and inefficacy in a man with recurrent thromboembolism, and anticoagulant‐associated priapism

Case studies in therapeutics: warfarin resistance and inefficacy in a man with recurrent thromboembolism, and anticoagulant‐associated priapism

Placental ecto-ATP diphosphohydrolase: its structural feature distinct from CD39, localization and inhibition on shear-induced platelet aggregation.

Human placental ecto-ATP diphosphohydrolase (ATPDase), an 82 kDa single-chain glycoprotein, was purified to high specific activity using a specific murine monoclonal antibody MK33 (IgG1-kappa). Structurally, protein-based analysis showed this enzyme to be almost identical to that of CD39 lymphoid cell activation antigen deduced by cDNA sequencing (Maliszewski CR, et al, J Immunol 1994; 153:3574); but differing in the NH2-terminal amino acid sequence, suggesting that placental ecto-ATPDase is most likely an isoform of CD39 generated by alternative splicing of the pre-mRNA. Functionally, placental ecto-ATPDase totally inhibits the secondary platelet aggregation induced by agonists at a final concentration (f.c.) of 1 microgram/ml. The purified enzyme (1 microgram/ml, final), pre-incubated with washed platelets prior to alpha-thrombin stimulation, completely inhibits the activation of platelet glycoprotein (GP) IIb/IIIa, thereby blocking the binding of fibrinogen or von Willebrand factor to platelets. Further, under different shear stresses, the enzyme modulates platelet aggregation differently. Low shear stress-induced platelet aggregation is blocked by this enzyme in a dose-dependent manner and is totally blocked at f.c. 0.5 microgram/ml. Under high shear stress, however, this protein at a f.c. of 0.5 microgram/ml mediates almost complete disaggregation of platelets without affecting the initial aggregation. Using immunohistochemical analysis, this enzyme was observed to be localized at the syncytiotrophoblasts of placental microvilli and the endothelial cells (ECs) of the umbilical vein obtained at full-term normal delivery, but scarcely at the ECs of the umbilical artery.

SIGNAL TRANSDUCTION SYSTEM IN EPINEPHRINE STIMULATED PLATELETS; COMPARISON BETWEEN EPINEPHRINE SENSITIVE AND INSENSITIVE PLATELETS

We recently reported the high prevalence of impaired platelet responsiveness only to epinephrine in healthy Japanese. This abnormality was associated with a 50% decrease in the number of α 2-adrenergic receptors. Platelets from non-responders (NR) do not undergo secondary platelet aggregation even after exposure to 100 μM epinephrine, but they can potentiate the effect of ADP to provoke platelet aggregation. To further define the nature of the defect and to delineate controversial steps of epinephrine stimulated signal transduction, a signaling pathway of epinephrine was investigated in platelets from NR and R(normal responder to epinephrine). In a unique particle counting apparatus, epinephrine initially triggered the formation of small platelet aggregates composing of 10–1000 cells from both R and NR, but the aggregates became larger (4600 > cells) only in platelets from R. Thus, platelets from NR lack the ability to form larger aggregates. A similar defect was reproduced by treating normal platelets with aspirin. In the presence of fibrinogen, platelets from NR lacked phospholipase A 2 activation, determined by arachidonic acid liberation in the presence of inhibitors to cyclooxygenase and lipoxygenase. In the absence of fibrinogen, aggregation and phospholipase A 2 activation were not evident in R and NR. The surface expression of GPIIb/IIIa was markedly decreased in platelets from NR after stimulation by epinephrine, in comparison with those from R. The resting level and epinephrine stimulated increase in cAMP were not significantly different between NR and R. Incubating R platelets with a half saturating dose of yohimbine rendered them insensitive to epinephrine. These results indicated that the impaired platelet aggregation induced by epinephrine was due to the impaired surface exposure of glycoproteins GPIIbIIIa integral to the activation of phospholipase A 2, which requires the full and normal occupancy of the α 2-adrenergic receptor by epinephrine. Copyright © 1997 Elsevier Science Ltd

Blood coagulation and platelet aggregation in major depression

Recently, it was found that the plasma of depressed patients significantly reduced the primary and secondary platelet aggregation to aggregating agents, such as ADP and collagen, in platelet rich plasma (PRP) of normal volunteers. Other authors found significantly decreased maximum amplitudes of adrenaline-induced platelet aggregation in major depressed patients versus normal controls. The aim of the present study was to examine platelet aggregation and blood coagulation in depression. Toward this end, the authors have measured secondary platelet aggregation to ADP and collagen, and the activated partial thromboplastin time (APTT) and prothrombin time (PT) in 16 normal volunteers, 16 minor, 40 simple major and 23 melancholic subjects. There were no significant differences in ADP- or collagen-induced platelet aggregability, APTT or PT between normal controls, minor, simple major or melancholic depressed patients. There were no significant relationships between severity of depression and APTT, PT or platelet aggregability to ADP or collagen. It is concluded that blood coagulation and platelet aggregability to ADP and collagen are probably not disordered in major depression.

The effect of inhaled anesthetics on the platelet aggregation and the ligand-binding affinity of the platelet thromboxane A2 receptor.

The mechanism by which anesthetics suppress platelet aggregation has not been elucidated. We determined the effects of halothane, enflurane, and isoflurane on human platelet aggregation induced by adenosine diphosphate (ADP), epinephrine, and a thromboxane A2 (TXA2) analog, and on ligand binding to the platelet TXA2 receptor. Halothane (2.6 mM) strongly suppressed ADP- and epinephrine-induced secondary aggregation of platelets, without significant alteration of primary aggregation. Platelet aggregation induced by a specific TXA2 agonist, (+)-9,11-epithia-11,12-methano-TXA2 (STA2), was suppressed by halothane, enflurane, and isoflurane in a concentration-dependent manner; the concentration of halothane, enflurane, and isoflurane which induced 50% inhibition (IC50) were 3.2, 12.3, and 15.7 mM, respectively (or 4.7, 9.8, and 24 minimum alveolar anesthetic concentration [MAC], respectively). The binding of a specific TXA2 receptor antagonist, 3H-S145, was significantly reduced by halothane (14-28 mM), but not by enflurane (20 mM) and isoflurane (20 mM). Scatchard analysis revealed that halothane (14 mM) increased Kd from 0.53 nM to 14.3 nM but did not alter Bmax significantly. These results indicate that halothane has a stronger suppressive effect on platelet aggregation than enflurane and isoflurane, and that the effect of halothane on platelet aggregation is due to reduction of the ligand-binding affinity of the platelet TXA2 receptor.

The Effect of Inhaled Anesthetics on the Platelet Aggregation and the Ligand-Binding Affinity of the Platelet Thromboxane A2 Receptor

The mechanism by which anesthetics suppress platelet aggregation has not been elucidated.We determined the effects of halothane, enflurane, and isoflurane on human platelet aggregation induced by adenosine diphosphate (ADP), epinephrine, and a thromboxane A2 (TXA2) analog, and on ligand binding to the platelet TXA (2) receptor. Halothane (2.6 mM) strongly suppressed ADP- and epinephrine-induced secondary aggregation of platelets, without significant alteration of primary aggregation. Platelet aggregation induced by a specific TXA2 agonist, (+)-9,11-epithia-11,12-methano-TXA (2) (STA2), was suppressed by halothane, enflurane, and isoflurane in a concentration-dependent manner; the concentration of halothane, enflurane, and isoflurane which induced 50% inhibition (IC50) were 3.2, 12.3, and 15.7 mM, respectively (or 4.7, 9.8, and 24 minimum alveolar anesthetic concentration [MAC], respectively). The binding of a specific TXA2 receptor antagonist,3 H-S145, was significantly reduced by halothane (14-28 mM), but not by enflurane (20 mM) and isoflurane (20 mM). Scatchard analysis revealed that halothane (14 mM) increased Kd from 0.53 nM to 14.3 nM but did not alter Bmax significantly. These results indicate that halothane has a stronger suppressive effect on platelet aggregation than enflurane and isoflurane, and that the effect of halothane on platelet aggregation is due to reduction of the ligand-binding affinity of the platelet TXA2 receptor. (Anesth Analg 1995;81:114-8)

Inhibition of platelet aggregation in whole blood by alcohol

Our previous studies have demonstrated that addition of moderate volumes of absolute alcohol (34 – 170 mM final concentration) to whole blood produces concentration-dependent platelet aggregation, due to release of adenosine diphosphate (ADP) from erythrocytes. We have now investigated the effects of exposure of blood to ethanol by a more “physiologic” protocol, in which 7.8% (w/v) alcohol is added to achieve a final concentration of 1 to 85 mM in human and rat blood or platelet rich plasma (PRP). The effects of short incubation with alcohol on platelet aggregation induced by ADP, collagen and arachidonic acid were examined by the impedance method of aggregometry. Aggregation induced by collagen in PRP of either species was significantly inhibited by 85 mM ethanol, with concentrations as low as 4.25 mM inhibiting the response to collagen in rat whole blood. ADP stimulated only primary, reversible aggregation in rat PRP and whole blood, and these responses were unaffected by alcohol. Human platelets responded to ADP with irreversible aggregation, which was significantly attenuated by 85 mM ethanol in whole blood but not PRP. Arachidonic acid evoked irreversible platelet aggregation in all four preparations; this was significantly inhibited by the high dose ethanol in human and rat PRP, but not whole blood. In contrast to our earlier studies with absolute ethanol, there was no evidence of hemolysis (and therefore, ADP release from red blood cells) using the current protocol. The results of these experiments show that alcohol, at physiologically relevant concentrations, has an inhibitory effect on secondary platelet aggregation responses to some agonists in whole blood as well as PRP, possibly by its previously demonstrated effects on arachidonic acid release by phospholipases. The possibility remains to be considered that other blood cells might contribute to the effects of alcohol on platelet aggregation in whole blood.

Antihistaminic drug bromadryl and stimulated platelet aggregation

The H1-receptor antagonist bromadryl dose-dependently inhibited stimulated rat platelet aggregation in vitro. Bromadryl was 10 times more effective on the secondary aggregation of thrombin-stimulated platelets compared with its inhibition of ADP-stimulated primary platelet aggregation in plasma. The inhibition of aggregation was accompanied by a dose-dependent inhibition of thrombin-stimulated malondialdehyde formation and thromboxane B2 production. The results indicate that bromadryl may interfere intracellularly with membrane phospholipid peroxidation and the arachidonic acid metabolism of stimulated platelets. Bromadryl, like other cationic amphiphilic drugs, may inhibit stimulated platelet functions by decreasing the stimulus-induced activation of phospholipase A2. Our results support the possible interference of bromadryl with histamine as an intraplatelet messenger responsible for aggregation.

Platelet-aggregation inhibiting activity of human placental chorioepithelial brush border membrane vesicles and basal plasma membrane vesicles

This laboratory investigated the platelet-aggregation inhibiting activity of human placental brush border membrane vesicles (BBMV) and basal plasma membrane vesicles (BpMV), and obtained the following results. o 1) A strong platelet-aggregation inhibiting activity exists in placental BBMW. In a 20 μg/ml protein concentration, the BBMV completely inhibited the secondary platelet aggregation induced by ADP. In a 40 μg/ml protein concentration, the BBMV completely inhibited the platelet aggregation induced by arachidonic acid and collagen, and in a 200 μg/ml protein concentration, the BBMV completely inhibited the platelet aggregation induced by ristocetin. However, placental BpMV did not show prominent platelet-aggregation inhibiting activity on platelet aggregation induced by ADP, arachidonic acid, collagen, or ristocetin. 2) A strong ADP degrading activity (ADPase activity) in the placental BBMV was found. ADP-degrading activity of the BBMV was 10.5±0.5 μmol/mg protein/minute and ADP-degrading activity of BpMV was 0.6±0.1 μmol/mg protein/minute. 3) The placental BBMV inhibited platelet thromboxane A 2 (TXA 2 ) production. In a 40 μg/ml protein concentration of placental BBMV, a platelet TXA 2 (TXB 2 ) production was almost completely inhibited. BpMV did not show prominent inhibitory activity on platelet TXA 2 (TXB 2 ) production. These results indicate that brush border membrane and basal plasma membrane have a different function in the respect of platelet-aggregation inhibiting activity, and that the brush border membrane may play an important role inhibiting platelet aggregation.