Whole Blood Platelet Research Articles

Effects of nitric oxide/EDRF on platelet surface glycoproteins.

We examined the effects of nitric oxide (NO)/endothelium-derived relaxing factor (EDRF) on platelet surface glycoproteins (GP). As determined by flow cytometry, in both a washed platelet system and platelet-rich plasma, the EDRF congener (S-nitroso-N-acetylcysteine) markedly inhibited both the thrombin-induced and the (stable thromboxane A2 analogue) U-46619-induced upregulation of P-selectin (alpha-granule protein), CD63 (lysosomal protein), and the GPIIb-IIIa complex (fibrinogen receptor) but minimally inhibited downregulation of the GPIb-IX complex (von Willebrand factor receptor). The inhibitory effects of EDRF were markedly reduced in whole blood or by the addition of washed erythrocytes. Platelets in whole blood were still responsive to guanosine 3',5'-cyclic monophosphate (cGMP), as shown by complete inhibition of P-selectin upregulation by the stable analogue N6,2'-O dibutyryl cGMP. These data suggests that 1) cGMP negatively regulates the platelet surface expression of P-selectin, CD63, and the GPIIb-IIIa complex but not the platelet surface expression of the GPIb-IX complex and 2) hemoglobin within erythrocytes inhibits the effects of EDRF/NO on platelet surface glycoproteins.

Inhibitory Effects of TA-993, a New 1,5-Benzothiazepine Derivative, on Platelet Aggregation

TA-993, an l-cis 4',8-dimethyl derivative of the Ca2+ antagonist diltiazem, and some of its metabolites inhibited platelet aggregation induced by collagen, ADP, epinephrine, platelet activating factor, arachidonic acid, and U-46619 in human platelets in vitro. Among the metabolites, MB3 was the most potent (IC50, <1 micromol/L; several hundred times more potent than the parent compound). The d isomer of MB3 was >100 times less potent than the l isomer. Unlike acetylsalicylic acid (ASA), TA-993 inhibited both primary and secondary phases of ADP-induced platelet aggregation and also exhibited a disaggregating effect on human platelet aggregates. The inhibitory effect of TA-993 was enhanced when used in combination with ASA. In ex vivo studies involving rats, TA-993 (approximately 0.3 to 100 mg/kg PO) dose-dependently inhibited collagen-induced platelet aggregation (ED50, 3 mg/kg PO). In the whole-blood platelet aggregation system in rats, orally administered TA-993 was also inhibitory in single (3 to 30 mg/kg) or repeated daily (10 mg/kg per day for 10 days) dosage. Orally administered TA-993 dose-dependently inhibited ADP-induced platelet aggregation ex vivo in dogs (0.3 to 10 mg/kg), significantly protected mice against collagen + epinephrine-induced thromboembolic death (10 mg/kg), and inhibited thrombus formation in an arteriovenous shunt in rats (30 mg/kg). The Ca2+-antagonistic action of TA-993 was very weak in depolarized canine basilar arteries: the potency was approximately 1/10 that of diltiazem (d-cis) and d-TA-993. These results suggest that antiplatelet action is more characteristic of the l-cis than the d-cis 1,5-benzothiazepine structure and that TA-993 may become a clinically useful antiplatelet agent of this structure series.

Effects of recombinant human megakaryocyte growth and development factor on platelet activation

The c-Mpl receptor for thrombopoietin and its recombinant related protein, the megakaryocyte growth and development factor (MGDF), is also expressed on circulating platelets. In the present study we evaluated the effect of MGDF on platelet aggregation in platelet-rich plasma (PRP) and in whole blood. The results obtained indicate that MGDF by itself did not affect platelet aggregation. However, when added before other agonists such as adenosine diphosphates (ADP), epinephrine (EPI), and thrombin (THR), it rendered platelets more sensitive. This "priming" effect of MGDF was dependent on the dose and on the time of platelet preincubation, and it occurred both in PRP and in whole-blood platelet aggregation. MGDF also "primed" the release of adenosine triphosphates and the production of thromboxane B2 by platelets stimulated with ADP, EPI, and THR. When added 15 seconds after the preincubation of platelets with subthreshold concentrations of ADP, EPI, and THR, MGDF exhibited a synergism with these agonists. Moreover, we observed a "priming" effect of MGDF on the phosphorylation of p-42 mitogen-activated protein kinase promoted by ADP, EPI, and THR. These observations suggest that thrombopoietin may play a physiologic role in modulating the response of platelets to several stimuli and thereby their hemostatic potential.

Analysis of the priming activity of lipids generated during routine storage of platelet concentrates

Compounds generated during the routine storage of platelet concentrates may have deleterious effects on the transfusion recipient. Daily plasma samples from platelet concentrates, both apheresis platelets and those separated from whole blood, were obtained serially during routine storage. These plasma samples were assayed for their ability to prime the NADPH oxidase in isolated human neutrophils. Quantitative and qualitative analysis of the priming agents was completed by lipid extraction, high-pressure liquid chromatography separation, and gas chromatography/mass spectroscopy. Compounds were generated in both apheresis and whole-blood platelets that significantly primed the NADPH oxidase after 24 and 48 hours of storage, respectively. The priming activity was maximal by component outdate: 2.6-fold that of the buffer-treated control neutrophils (apheresis) and 3.9-fold that of the buffer-treated control neutrophils (whole blood). These agents were generated by cellular constituents, as stored plasma did not demonstrate such priming activity. Inhibition of this priming activity by WEB 2170, a specific platelet-activating factor receptor antagonist, suggested that the observed priming involved the platelet-activating factor receptor. A portion of the priming activity from platelet concentrates was organically extractable: 69 percent of that from apheresis platelets and 46 percent of that from whole-blood platelets. Further purification of the lipid's priming activity by normal-phase high-pressure liquid chromatography demonstrated a single peak of priming activity at the retention time of lysophosphatidylcholines. Because 46 percent of the priming activity from whole-blood platelets was chloroform insoluble and because it has been reported that interleukin 8 is generated during routine storage of whole-blood platelets, the effects of interleukin 8 on the NADPH oxidase were examined. Recombinant monocyte interleukin 8 rapidly primed the oxidase but was not inhibited by WEB 2170. Lipids were generated during the routine storage of platelet concentrates that prime the NADPH oxidase, and they may play a role in the severe complications of transfusion therapy. Other non-lipid compounds, such as interleukin 8, that are generated in whole-blood platelets may also contribute to the observed priming activity of plasma.

Rapid flow cytometric quantitation of reticulated platelets in whole blood

Young or reticulated platelets contain some residual mRNA, which is rapidly degraded after platelet release into the circulation. In order to minimize platelet activation and possible loss of large platelets during sample handling a whole blood method has been developed utilising the RNA fluorochrome thiazole orange (TO) in combination with an antibody to anti-glycoprotein Ib (GpIb) directly conjugated to phycoerythrin (PE), to specifically stain reticulated platelets via flow cytometric analysis. In this study whole blood analysis of platelet mRNA was undertaken in healthy normal subjects and a variety of patients with haematological abnormalities. The percentage of Gp Ib positive platelets containing mRNA in normals (n = 22) was 11.61% with a two SD range of 3.19-20.01%. The percentage of reticulated platelets was significantly increased (mean mRNA content ± one SD) in sickle cell disorders (n = 22) 38.12% ± 18.42 (P < 0.001); thalassaemia (major, intermedia and trait) (n = 24) 29.76 ± 19.15 (P < 0.001); ITP (N = 20) 23.53% ± 13.04 (P < 0.02) and essentialthrombocythemia (N = 32) 37.12 ± 19.84 (P < 0.001). Platelets from patients with reactive thrombocytosis (N = 15) were only 12.23% positive (±6.95) and not significantly different from the normal range (P = 0.95). This method offers a rapid and simple procedure for assessment of reticulated platelets in whole blood and suggests that there may be an increased platelet turnover in certain haemoglobinopathies.

Hormone replacement therapy reduces the reactivity of monocytes and platelets in whole blood — A beneficial effect on atherogenesis and thrombus formation?

OBJECTIVE: Our purpose was to investigate the effects of hormone replacement therapy on the reactivity of monocytes and platelets in whole blood, measured by tissue factor activity, tumor necrosis factor-α, and thromboxane B 2. STUDY DESIGN: Thirty-two women were randomized into either transdermal or oral combined hormone replacement therapy and underwent blood sampling before and after 3 and 12 months of treatment. The tissue factor activity in monocytes was measured both in unstimulated whole blood and after a weak lipopolysaccharide stimulation. Tumor necrosis factor-α and thromboxane B 2 formation in plasma were measured after a weak lipopolysaccharide stimulation of whole blood. RESULTS: After 12 months of hormone replacement therapy there were significant reductions of tissue factor activity in both unstimulated and lipopolysaccharide-stimulated monocytes ( p < 0.001) and significant reductions in the formation of tumor necrosis factor-α ( p < 0.03) and thromboxane B 2 ( p < 0.02). There were no differences in these parameters between the transdermal and the oral groups. No changes were observed after 3 months of therapy. CONCLUSION: Twelve months of hormone replacement therapy reduces cellular activation of blood monocytes and platelets; these changes may account for some of the beneficial effects in reducing the risk of cardiovascular disease.

A comparative study of whole-blood platelet aggregation in laboratory animals: its species differences and comparison with turbidimetric method

We undertook a systematic comparison of whole-blood platelet aggregation concerning species difference of commonly used laboratory animals (rabbits, guinea pigs, rats and dogs), agonist difference (collagen, ADP and platelet activating factor [PAF] and technical difference against a traditional method of turbidimetry. Collagen-induced aggregation with whole-blood aggregometry was less variable among species tested and was similar to the results obtained by turbidimetry, which measures platelet aggregation in citrated plasma. In contrast, whole-blood aggregometry in responses to ADP and PAF showed marked species difference, being different from turbidimetry. Our results provide species difference among laboratory animals and suggest that the modulations of platelet aggregation by other blood elements probably differ in each species and with stimulating agonists.

Physical and chemical effects of red cells in the shear-induced aggregation of human platelets

Both chemical and physical effects of red cells have been implicated in the spontaneous aggregation of platelets in sheared whole blood (WB). To determine whether the chemical effect is due to ADP leaking from the red cells, a previously described technique for measuring the concentration and size of single platelets and aggregates was used to study the shear-induced aggregation of platelets in WB flowing through 1.19-mm-diameter polyethylene tubing in the presence and absence of the ADP scavenger enzyme system phosphocreatine-creatine phosphokinase (CP-CPK). Significant spontaneous aggregation was observed at mean tube shear rates, (G) = 41.9 and 335 s-1 (42% and 13% decrease in single platelets after a mean transit time (t) = 43 s, compared to 89 and 95% decrease with 0.2 microM ADP). The addition of CP-CPK, either at the time of, or 30 min before each run, completely abolished aggregation. In the presence of 0.2 microM ADP, CP-CPK caused a reversal of aggregation at (t) = 17 s after 30% of single cells had aggregated. To determine whether red cells exert a physical effect by increasing the time of interaction of two colliding platelets (thereby increasing the proportion of collisions resulting in the formation of aggregates), an optically transparent suspension of 40% reconstituted red cell ghosts in serum containing 2.5-micron-diameter latex spheres (3 x 10(5)/microliters) flowing through 100-microns-diameter tubes was used as a model of platelets in blood, and the results were compared with those obtained in a control suspension of latex spheres in serum alone. Two-body collisions between microspheres in the interior of the flowing ghost cell or serum suspensions at shear rates from 5 to 90 s-1 were recorded on cine film. The films were subsequently analyzed, and the measured doublet lifetime, tau meas, was compared with that predicted by theory in the absence of interactions with other particles, tau theor. The mean (tau meas/tau theor) for doublets in ghost cell suspensions was 1.614 +/- 1.795 (SD; n = 320), compared to a value of 1.001 +/- 0.312 (n = 90) for doublets in serum. Whereas 11% of doublets in ghost cell suspensions had lifetimes from 2.5 to 5 times greater than predicted, in serum, no doublets had lifetimes greater than 1.91 times that predicted. There was no statistically significant correlation between tau meas/tau theor and shear rate, but the values of tau meas/tau theor for low-angle collisions in ghost cell suspensions were significantly greater than for high-angle collisions.

Release of Platelet Plasminogen Activator Inhibitor 1 in Whole Blood Is Increased in Patients With Type II Diabetes

To compare platelet plasminogen activator inhibitor 1 (PAI-1) release in type II diabetic patients and healthy control subjects. We studied a group of 27 diabetic patients and a group of 16 nondiabetic control subjects. Whole-blood platelet aggregation, defined as a decrease in platelet count during shaking (180 rpm) of blood samples at 37 degrees C, and plasma PAI-1 antigen concentrations were measured in parallel at time 0, 7.5, 15, 30, 60, 120, and 180 min. Platelet aggregation did not differ significantly between the two groups at any time period. However, the increase in plasma PAI-1 antigen concentration over basal levels at time 0 was higher for the group of diabetic patients when compared with their matched control subjects. The increment of PAI-1 antigen was 61.8 +/- 29.4 vs. 35.9 +/- 13.4 ng/ml (P < 0.005, means +/- SD) after 180 min for the diabetic and control subjects, respectively. Platelet PAI-1 release was correlated to very-low-density lipoprotein cholesterol and triglyceride plasma levels, but not to HbA1c levels. Platelets of patients with type II diabetes release significantly more PAI-1 than platelets of healthy subjects at the same level of platelet aggregation. This may contribute to enhanced thrombosis in diabetes.

Whole-Blood Platelet Aggregation Predicts In Vitro and In Vivo Primary Hemostatic Function in the Elderly

Increased platelet aggregation is associated with higher coronary artery disease mortality. Enhanced platelet aggregation in platelet-rich plasma has also been described in the elderly. To define age-related changes in primary hemostasis, we studied 37 elderly and 31 young blood donors. There were no significant age-related differences in whole-blood platelet aggregation, platelet adherence and thrombus formation on human umbilical artery segments, or bleeding time. Plasma fibrinogen was significantly higher in elderly men and women, whereas activated factor VII was elevated only in elderly women. Collagen-induced platelet aggregation was significantly correlated with platelet adherence to the subendothelium in elderly (r = .488, P = .002) but not in young donors. Accordingly, collagen-induced platelet aggregation showed a significant inverse correlation with bleeding time only in the elderly (r = -.401, P = .014). Arachidonic acid-induced platelet aggregation was significantly associated with platelet adherence to the subendothelium (r = .658, P = .003) and bleeding time (r = -.540, P = .021) only in elderly men. In young donors, ADP-induced platelet aggregation was significantly correlated with platelet adherence to the thrombogenic adventitial surface (r = .395, P = .031); in the elderly this association only approached significance (r = .315, P = .058). Whole-blood platelet aggregation in response to collagen and arachidonic acid may be more useful in predicting primary hemostatic function in the elderly than in the young. Furthermore, in the elderly, the correlation between platelet aggregation in whole blood and platelet-arterial wall interactions in vitro and in vivo may contribute to the ability of this test to predict coronary risk.

The Platelet Aggregation Score and Its Correlation to β- Thromboglobulin and Platelet Factor 4 Levels

We describe a new method of interpreting platelet aggregometry results and its correlation to platelet secretory protein levels. We studied 123 healthy male volunteers, 37-85 years of age. The aggregation response to 2.5 μM ADP was measured in a standardized platelet suspension (SPS) prepared from platelet-rich plasma. Turbidimetic measurements included amplitude of the primary aggregation wave (Amp1), maximum aggregation amplitude (Amax), and area under the aggregation curve (Sagg). A platelet aggregation (PA) score was calculated as ( Amp1 /Amax) ·Sagg ·(whole-blood platelet count/SPS platelet count). β-Thromboglobulin (β-TG)and . platelet factor 4 (PF4) levels were measured by radioimmunoassay. The PA score correlated significantly (p &lt; 0.0001) with β-TG ( r = 0.86) and PF4 ( r = 0.87). It was concluded that the PA score correlates closely with individual subjects' propensities to platelet activation, as reflected by plasma β-TG and PF4 levels. Key Words: Platelets-Platelet aggregometry-Platelet secretory proteins.

931-112 A Low-dose Intravenous Aspirin, Bolus Injection, Effectively Inhibits Platelet Aggregation

Prompt inhibition of platelet aggregation is important in acute coronary syndrome and before an intervention procedure. To determine whether a single low dose of iv aspirin inhibits platelet aggregation, twenty-seven healthy volunteers (7 F and 20 M), mean age 43.5 years, were randomized double-blind to a single iv low dose of aspirin DL-Iysine (L-ASA) equivalent to 2 mg/kg of aspirin, high dose (H-ASA) equivalent to 10 mg/kg, or placebo (PI). Platelet aggregation were performed before and 1 hand 24 h after drug administration, in whole blood (WB) using electrical impedance and in platelet-rich plasma (PRP) by optical light transmittance. Baseline WB platelet aggregation (Col 3 μg/mI) was the same with L-ASA, H-ASA and PI (24 ± 5, 23 ± 3 and 24 ± 4rl respectively) and decreased significantly more with L-ASA and H-ASA than with PI after 1 h.(17 ± 6, 15 ± 7 and 21 ± 5Ω p &lt; 0.0l)and 24 h(17 ± 7, 16 ± 6 and 25 ± 4Ω P &lt; 0.01). in PRP were similar: PR Poptical aggregation (%) Before AfterASA 1 h. 24 h ADP (5/LM): L-ASA 73 ± 21 50 ± 14 * 45 ± 15 * H-ASA 65 ± 23 54 ± 17 * 52 ± 16 * PI 69 × 18 72 ± 22 69 ± 14 Col (3 /Lg/ml): L-ASA 64 ± 19 33 ± 13 * 36 ± 18 * H-ASA 60 ± 16 32 ± 17 * 34 ± 14 * PI 55 ± 13 56 ± 18 57 ± 14 * p &lt; 0.001 (ANOVA test) respect to PI and the baseline values No differences in response were observed between the two doses of aspirin and no significant changes occurred between 1 and 24 hours in any group. Effective inhibition of platelet aggregation is thus achieved within 1 hour after the administration of low-dose 2 mg/kg of ivaspirin.

White cell-associated procoagulant activity induced by ABO incompatibility.

Disseminated intravascular coagulation is an established complication of acute hemolytic transfusion reactions, particularly those involving the ABO red cell (RBC) antigen system. In addition, peripheral blood white cells, particularly monocytes, have demonstrated expression of procoagulant activity (PCA) in response to inflammatory stimuli. To better define the activation of coagulation in immune hemolysis, in vitro experiments were conducted to investigate the expression of PCA by peripheral blood white cells in ABO RBC incompatibility. Fresh group O heparinized whole blood was incubated with washed, packed group A or O RBCs. White cells were separated, washed, and lysed before assay of PCA, which was measured by a one-stage recalcified clotting time assay. Units of activity were calculated on the basis of a rabbit brain thromboplastin standard curve. Mechanisms of coagulation activation were investigated by using specific coagulation factor-deficient plasmas, blocking antibodies to tissue factor, and anti-CD11b. Significant levels of white cell-associated PCA were found at 2 to 6 hours in response to incompatible (group A) RBCs, but not in response to compatible (group O) RBCs. PCA was not correlated with numbers of platelets in whole blood. Nonimmune lysis of compatible RBCs did not induce PCA. When whole blood reconstituted from washed cells and heat-inactivated plasma was incubated with incompatible RBCs, PCA and hemolysis were abrogated, which suggests that complement is a required intermediate. Protein synthesis inhibition by the addition of cycloheximide (5 mg/mL) to whole blood incubated with RBCs prevented the expression of PCA. Substitution of factor VIII-deficient plasma for normal plasma in the recalcified clotting time assay had no effect, whereas PCA was reduced by 68 percent with factor VII-deficient plasma and was unmeasurable with factor X-deficient plasma. PCA was restored by a 1-to-1 mix of normal and factor VII-deficient plasma. Incubation of samples in the PCA assay with tissue factor antibodies resulted in up to 86-percent inhibition of measured PCA. Titration of the response to the amount of tissue factor antibodies added demonstrated that maximal inhibition occurred with 0.45 mg per mL, above which no further inhibition took place. However, the addition of anti-CD11b (0.75 mg/mL) concomitantly with anti-tissue factor abolished measurable activity. This effect was independent of the amount of added protein, and anti-CD11b alone had no effect on measured activity. The addition to whole blood concomitantly with RBCs of polyclonal antibodies to tumor necrosis factor, sufficient to neutralize 2000 pg per mL, did not alter PCA expression. These results indicate that white cell-associated PCA is generated in whole blood in response to ABO RBC incompatibility and may contribute to disseminated intravascular coagulation in acute hemolytic transfusion reactions. Two possible cellular mechanisms are suggested, which involve tissue factor expression and the activation of factor X by a CD11b-dependent mechanism.

N,N,N-trimethylsphingosine modifies aggregatory response and ATP release from platelets in whole blood

We investigated the influence of N,N,N-trimethylsphingosine (TMS), a potent inhibitor of protein kinase C (PKC), on whole blood aggregation and ATP release from platelets. The preincubation with TMS at 1μM for 2 min enhanced ATP release during arachidonic acid-induced aggregation. The ristocetin-induced agglutination was inhibited in a TMS concentration-dependent manner, which suggests that TMS may interact with the vWF receptor on platelets. TMS suppressed aggregatory response and ATP release from platelets after the addition of collagen. In contrast, the platelet aggregation induced by ADP and 12-O-tetradecanoylphorbol-13-acetate (TPA) was only slightly inhibited and the ATP release was not influenced after preincubation with TMS. Our results are in contrast to previous reported data, which were obtained using PRP and washed platelets.

Whole-blood platelet aggregation, buccal mucosa bleeding time, and serum cephalothin concentration in dogs receiving a presurgical antibiotic protocol

Summary Whole-blood platelet aggregation (using the impedance method) and adenosine triphosphate (atp) release, buccal mucosal bleeding time (bt), and serum cephalothin concentration were measured in 21 adult female Beagles before (pre) and 1 hour (1hr) after iv administration of cephalothin (22 mg/kg). A second injection of cephalothin (22 mg/kg) was given 3 hours after the first, and blood samples were obtained 1 hour (4 hr, 4 hours after the first injection) and 3 hours (6 hr, 6 hours after the first injection) after the second injection. Samples of jugular blood were obtained from each dog, using citrate as an anticoagulant. A platelet count was obtained for each sample. Platelet aggregation and atp released from the aggregating platelets were measured within 1 hour of sample collection, using a whole-blood aggregometer. Adenosine diphosphate (ADP) and collagen were used as aggregating agents. Aggregation was measured over 6 minutes for each aggregating agent; atp release in response to collagen, but not to ADP, was measured over the same period. For 1 hr samples, there was a significant (P &lt; 0.01) reduction from pre values in the ability of platelets to aggregate in response to ADP. Bleeding time was determined, using a published procedure, with each dog as its own control. Bleeding time during the same period was found to be significantly increased over pre values for 1 hr (P &lt; 0.01) and 6 hr (P &lt; 0.02) samples. There was no significant difference between bt for 1 hr and 4 hr samples. Median serum cephalothin concentrations in pre, 1 hr,4hr, and 6 hr samples did not correlate significantly to platelet aggregation, atp release, platelet count, or bt at any time during the study. Because serum cephalothin concentration was so low, and because there are few reports of the kinetics of cephalothin after iv administration, serum cephalothin was measured in 2 dogs over 2 hours after single iv administration of cephalothin. Values reached a peak at 2 minutes after drug administration, then decreased rapidly over 10 to 15 minutes, and were low by 2 hours.

Detection of activated feline platelets in platelet-rich plasma by use of fluorescein-labeled antibodies and flow cytometry.

Platelets contribute to prethrombotic or thrombotic states; however, accepted evaluation methods (i.e., in vitro testing by use of an aggregometer) of platelet function in cats can be difficult because of the large volume of blood required from which platelets are isolated and the potential for platelet activation due to difficult venipunctures in sometimes uncooperative or excited animals. The activation problem also contributes to errors in platelet counts. Platelets from four domestic short haired cats (two males, two females, 2-3 years old) minimally restrained without sedation or anesthesia were evaluated. Blood (5 ml) was collected by jugular venipuncture directly into syringes containing 3.8% trisodium citrate (nine parts blood to one part anticoagulant) plus prostaglandin E1 (3 microM; 0.25, 0.5, 1, or 2 microliters/500 microliters citrate) or 3.8% trisodium citrate alone. Prostaglandin E1, which is a stable metabolite of arachidonic acid with platelet inhibitory properties similar to those of prostaglandin I2, was added to the anticoagulant to prevent activation of platelets during the collection process. Feline platelets exposed to prostaglandin E1 became immediately and persistently nonreactive to agonists, which negated their use in functional studies (aggregation, 14C-serotonin release, binding of fluorescein-conjugated antifibrinogen) but improved platelet counting accuracy. Detection of in vivo activation of platelets in prethrombotic and thrombotic states in humans has been done by identification of activation-dependent molecules on platelet surfaces by use of specific antibody recognition and detection by flow cytometric analysis. Many activation-dependent platelet surface receptor changes are species specific; however, fibrinogen appears to be conserved across species.(ABSTRACT TRUNCATED AT 250 WORDS)

Valproate-mediated disturbances of hemostasis: relationship to dose and plasma concentration.

Valproic acid (VPA) may cause impaired platelet function, thrombocytopenia and, occasionally, severe bleeding. Controversy exists both as to the mechanism of this alteration in hemostasis and as to whether these adverse effects are either dose-related or idiosyncratic. Previous investigations have focused primarily on pediatric patients who commonly were receiving multiple anticonvulsant medications. We evaluated a cohort of 27 adult patients with epilepsy who were receiving VPA monotherapy and compared them with age-matched controls to determine whether a correlation exists between platelet count, function, bleeding time, levels of von Willebrand's factor antigen, and VPA dose or plasma concentration. VPA patients had significantly lower platelet counts and longer bleeding times than did controls (p < 0.05). Platelet count was inversely correlated to VPA dose and both free and total VPA concentration (p < 0.01). There was no significant difference in levels of von Willebrand's factor antigen between patients and controls. We assessed platelet aggregation by measurement of whole-blood platelet aggregation and release with agonists including adenosine diphosphate, thrombin, collagen, arachidonic acid, and ristocetin. VPA patients had significant decreases in platelet aggregation values compared with controls. There were significant differences in collagen, arachidonic acid, and adenosine diphosphate release and aggregation between groups that correlated to both VPA dose (p < 0.01) and concentration (p < 0.05). Prolongation of bleeding time was significantly correlated to both VPA dose and concentration. Our data suggest a significant relationship between impaired platelet function and both VPA dose and plasma concentration.

Granule secretion markers on fluid-phase platelets in whole blood perfused through capillary tubing.

The effect of material composition and shear rate on fluid-phase platelet activation was investigated using a capillary perfusion model. Citrated whole blood was perfused along the lumens of tubes constructed from silicone, PVC, Pellethane, W124 (an experimental polyetherurethane), and glass. Platelet activation was determined by measuring the increase in alpha-granule membrane protein P-selectin (GMP-140, CD62) and the lysosomal granule membrane protein GP-53 (CD63) on fluid-phase platelets by flow cytometry. All tubes caused an increase over the negative control in the number of P-selectin and GP-53 molecules detectable on the surface of these platelets. The activation response of platelets to changes in shear rate was also investigated. It was found that lysosomal release paralleled alpha-granule release in glass, but not in Pellethane, over a range of wall shear rates (100-1,000 s-1).

Investigations on Spontaneous Aggregation and Platelet Responses to Streptokinase and to Adrenaline During Pregnancy

Platelets have been shown to be hyperresponsive during pregnancy. Studies in whole blood have revealed increased 'spontaneous' platelet aggregation (SPA) and increased aggregation and (14)C-5HT release in response to adrenaline. Here we have extended previous studies. We have explored the possibility that the nature of the agent used to anticoagulate the blood may have influenced the results obtained, and, for the first time, have investigated the effects of streptokinase (SK) on platelets in whole blood during pregnancy. We found that increased SPA is present during pregnancy irrespective of the anticoagulant used. Also, platelets in blood taken during pregnancy aggregate more extensively in response to adrenaline, even though the type of anticoagulant influences the extent of (14)C-5HT release that accompanies the aggregation response. SK was found to induce platelet aggregation in whole blood and this was also independent of the anticoagulant used. Further, SK induced aggregation at a lower concentration than in blood from non-pregnant female volunteers (NFV). Increased platelet responses had always returned to values similar to those for NFV by 12 weeks post-natal. These studies confirm the existance of a generalized increase in platelet reactivity during pregnancy, indicate that this is not an artefact consequent to the use of a particular anticoagulant, and provide new information on the effects of SK on platelets during pregnancy.

Reversible Inhibition of Human Platelet Activation by Hypothermia In Vivo and In Vitro

A hypothermia-induced hemorrhagic diathesis is associated with cardiopulmonary bypass, major surgery, and multiple trauma, but its pathophysiological basis is not well understood. We examined the hypothesis that hypothermia reversibly inhibits human platelet activation in vitro and in vivo. Platelet activation was studied in normal volunteers by whole blood flow cytometric analysis of modulation of platelet surface GMP-140 and the glycoprotein (GP) Ib-IX complex in: a) shed blood emerging from a standardized in vivo bleeding time wound; b) peripheral blood activated in vitro with either thrombin (in the presence of gly-pro-arg-pro, an inhibitor of fibrin polymerization) or the stable thromboxane (TX) A2 analogue U46619. Platelets in peripheral whole blood were activated at temperatures between 22 degrees C and 37 degrees C. the forearm skin temperature was maintained at temperatures between 22 degrees C and 37 degrees C prior to and during the bleeding time incision. Platelet aggregation was studied in shed blood by flow cytometry and in peripheral blood by aggregometry. Generation of TXB2 (the stable metabolite of TXA2) was determined by radioimmunoassay. In vitro, hypothermia inhibited both thrombin- and U46619-induced upregulation of GMP-140, downregulation of the GPIb-IX complex, platelet aggregation, and TXB2 generation. These inhibitory effects of hypothermia were all completely reversed by rewarming the blood to 37 degrees C. In vivo, platelet activation was inhibited by hypothermia as shown by 5 independent assays of shed blood: upregulation of GMP-140, downregulation of the GPIb-IX complex, platelet aggregate formation, TXB2 generation, and the bleeding time.(ABSTRACT TRUNCATED AT 250 WORDS)