Whole blood platelet aggregation in humans and animals: a comparative study

BACKGROUND. Many animal species are used to evaluate the performance and blood compatibility of cardiovascular devices, but interspecies differences in platelet activity have not been well characterized. This study measures platelet response to six agonists in human, dog, and calf blood. MATERIALS AND METHODS. We used whole blood impedance lumi-aggregometry to measure platelet aggregation and ATP release in blood samples from adult humans (n = 19), mongrel dogs (n = 19), and Holstein calves (n = 7). The agonists were collagen, ristocetin, arachidonic acid, thrombin, and three concentrations of both ADP and epinephrine. RESULTS. Only collagen (1 microg/ml) and ADP (5, 10, and 20 microM) caused aggregation and ATP release in all samples. Canine platelets responded to all six agonists at all doses. Human platelets responded to everything except epinephrine at 2 and 100 microM. Bovine platelets responded only to collagen, ADP, and thrombin. In bovine platelets, aggregation from collagen and ATP release from thrombin were significantly lower than the corresponding responses in human and canine blood. The aggregation induced by 10 microM ADP was significantly higher in canine than in human platelets. CONCLUSION. Human, canine, and bovine platelets have very different responses to agonists. In these models, collagen (1 microg/ml) and ADP (10 microM) are the agonists of choice for investigating whole blood platelet aggregation because they provide the most consistent results between species. For ATP release, 1 U/ml thrombin is the recommended agonist and the dose for all three species.     

Platelet aggregation in humans and nonhuman primates: relevance to xenotransplantation

Iwase H, Ekser B, Zhou H, Dons EM, Cooper DKC, Ezzelarab MB. Platelet aggregation in humans and nonhuman primates: relevance to xenotransplantation. Xenotransplantation 2012; 19: 233–243.. © 2012 John Wiley & Sons A/S. Abstract: Introduction: Platelet activation/aggregation plays a key role in the dysregulation of coagulation and the development of thrombotic microangiopathy in nonhuman primate recipients of pig xenografts. As a preliminary to the study of anti‐platelet therapy in vitro and in vivo, the present study aimed to compare platelet aggregation in whole blood from humans, baboons, and cynomolgus monkeys. Methods: Using “Chrono‐log” technology (two‐sample four‐channel Chrono‐log Whole Blood Aggregometer), we studied aggregation of platelets in healthy humans (n = 8), baboons (n = 5), and monkeys (n = 8). Whole blood (WB) samples were collected, and platelet aggregation was assessed using three different volumes of blood (1, 0.5, and 0.25 ml). Platelet activation was induced using collagen (at 3 and 5 μg/ml), ristocetin (at 0.5 and 1.0 mg/ml), adenosine diphosphate (ADP; at 10, 20, and 40 μm ), or thrombin (at 1 and 5 IU/ml). Inhibition of agonist‐induced platelet aggregation by heparin and low molecular weight heparin (LMWH) (at 1, 10, and 100 IU/ml) was evaluated. Results: Mean platelet counts were 222.1, 263.2, and 276.1 (×10³/μl) in humans, baboons, and monkeys, respectively. In all three species, platelet aggregation was induced by collagen, ristocetin, ADP, or thrombin in a dose‐dependent manner. A blood volume of 0.5 ml provided the most consistent results with all agonists in all three species. Dilution studies indicated that there was a significant positive correlation between platelet count and percent aggregation of platelets (P < 0.05). Collagen (3 and 5 μg/ml), ADP (10, 20, and 40 μm ), and thrombin (1 and 5 IU/ml) induced significantly greater platelet aggregation in humans than in baboons. ADP (20 and 40 μm ) and thrombin (1 and 5 IU/ml) induced significantly greater platelet aggregation in monkeys than in baboons. There was no species difference with ristocetin (0.5 or 1.0 mg/ml). In all species, thrombin (1 or 5 IU) induced greater platelet aggregation than any of the other reagents. Heparin at 1 IU/ml and LMWH at 10 IU/ml in all species almost completely abrogated thrombin‐induced platelet aggregation. Heparin at 100 IU/ml effectively inhibited platelet aggregation induced by collagen, but only partially inhibited aggregation induced by ADP or ristocetin. LMWH only partially inhibited aggregation induced by collagen, ristocetin, and ADP. Conclusions: The “Chrono‐log” technology proved to be a reliable method of evaluating platelet activation and aggregation in vitro in primates. Species differences may play a role in platelet aggregation, with the monkey being more comparable to the human than the baboon, although overall trends were similar. In all species, thrombin induced greater platelet aggregation than other agonists. Even a concentration of heparin of 1 IU/ml, which is probably the maximal concentration that is clinically‐applicable, prevented platelet aggregation induced by thrombin, but was less effective in preventing aggregation induced by collagen, ADP, or, particularly, ristocetin.     

Clopidogrel withdrawal is associated with proinflammatory and prothrombotic effects in patients with diabetes and coronary artery disease

Inhibition of the P2Y12 pathway by the platelet antagonist clopidogrel is associated with a marked reduction in platelet reactivity. Recent reports have shown that P2Y12 inhibition has anti-inflammatory effects as well. However, whether clopidogrel withdrawal is associated with proaggregatory and proinflammatory effects has not yet been explored. Since diabetic subjects are characterized by a prothrombotic and proinflammatory status, we hypothesize that these patients may be more vulnerable to these effects. A total 54 patients with diabetes on long-term (12 months) dual antiplatelet therapy (aspirin plus clopidogrel) were studied. Platelet aggregation (following 6 and 20 micromol/l ADP stimuli) and inflammatory markers (C-reactive protein and P-selectin expression) were assessed before and 1 month following clopidogrel withdrawal. Following clopidogrel withdrawal, aspirin responsiveness using platelet function analyzer-100 was determined as well. A significant increase in all the assessed platelet (P < 0.0001 for 6 and 20 micromol/l ADP-induced aggregation) and inflammatory (P < 0.05 for C-reactive protein, P < 0.001 for P-selectin expression in resting platelets, and P < 0.0001 for P-selectin expression in ADP-stimulated platelets) biomarkers was observed following clopidogrel withdrawal. Low responders to aspirin had increased platelet aggregation profiles (P < 0.05 for 6 and 20 micromol/l ADP-induced aggregation) but no differences in inflammatory markers. In conclusion, clopidogrel withdrawal is associated with an increase in platelet and inflammatory biomarkers in diabetic patients, supporting pleiotropic effects coupled with P2Y12 receptor antagonism.     

Flow cytometric assessment of platelet function in patients with peripheral arterial occlusive disease.

This study compared new and traditional measures of platelet function in 16 patients with severe peripheral arterial occlusive disease and 15 age-matched controls. Circulating platelets were characterized by the use of fluorescence flow cytometry to assess platelet aggregate formation and expression of the secretion-dependent alpha granule membrane protein GMP-140, by measurement of plasma beta-thromboglobulin (beta-TG), and by performance of platelet-rich plasma aggregation studies. In addition, blood samples were treated with graded concentrations of adenosine diphosphate (ADP; 0 to 10 mumols/L) to characterize by fluorescence flow cytometry the secretory and aggregatory responses to mild stimulation. No differences were detected between the two groups with regard to platelet function in unstimulated circulating blood by use of these techniques. Values (mean +/- SEM) observed were: GMP-140-positive platelets, 11% +/- 3% versus 13% +/- 2%; platelet aggregates in circulating whole blood, 4% +/- 1% versus 9% +/- 3%; plasma beta-TG, 92 +/- 12 versus 94 +/- 22 ng/ml; and ED50 (concentration of ADP required to produce half maximal aggregation), 3.8 +/- 1.1 versus 3.1 +/- 0.5 mumol/L in the patients with peripheral arterial occlusive disease and controls, respectively. Treatment with ADP caused a dose-related increase in GMP-140 expression in both groups, without significant differences in this parameter between the groups at any given concentration. However, stimulation with ADP concentrations greater than 1 mumol/L resulted in more frequent aggregate formation in the control than in the peripheral arterial occlusive disease group (25% +/- 4% versus 11% +/- 2%, respectively at 5.0 mumols/L, p = 0.002).(ABSTRACT TRUNCATED AT 250 WORDS)     

The influence of epidural analgesia on platelet function and correlation with plasma bupivacaine concentrations

The effect of epidural anaesthesia with bupivacaine 0.5% on platelet aggregation was studied in seven patients undergoing transurethral resection of the prostate. Peak plasma concentrations of bupivacaine 470 +/- 270 ng ml-1 occurred at 30 min after administration. At that time there were no significant changes in platelet aggregation. However, the maximum rate of the primary- and secondary-aggregation velocities induced by 1.0 microM ADP were significantly decreased at 1 h and 3 h after bupivacaine administration. The maximum percentage ADP-induced platelet aggregation was also decreased significantly at 1 h and 3 h. The minimum concentration of ADP required to induce secondary-phase platelet aggregation was significantly increased at 1 h but not at 3 h. There was a significant correlation between bupivacaine concentrations and all platelet aggregation parameters except the maximum ADP-induced aggregation. Platelet inhibition occurred at plasma bupivacaine concentrations that were considerably lower than those needed to produce similar inhibition in vitro.     

The effects of local anesthetics on maternal and neonatal platelet function

The effects of bupivacaine (B), lidocaine (L) and 2-chloroprocaine (C) on maternal (M) and neonatal (N) platelet function were studied using in vitro beta-thromboglobulin (beta-tg) release (radioimmunoassay), and in vitro platelet aggregation. Aggregation produced by adenosine diphosphate (ADP), epinephrine and collagen was measured in the presence of 1, 10, 100, 500 or 1000 micrograms/ml concentrations of B, L or C. In addition, spontaneous in vivo beta-tg release was measured in M and N blood. In vivo beta-tg level in M and N blood was approximately double that in non-pregnant subjects (p less than 0.025). In vitro beta-tg release in M and N samples was inhibited only at concentrations exceeding 1000 micrograms/ml, and the inhibition was less in M and N samples than in non-pregnant subjects. None of the anesthetics inhibited aggregation of M or N platelets at 1 and 10 micrograms/ml. Only concentrations of 500 micrograms/ml or greater consistently inhibited platelet aggregation produced by the three aggregants in M and N samples, and L was the least effective of the three agents. Neonatal platelet aggregation was affected more by local anesthetics than was maternal aggregation. It is concluded that plasma local anesthetic concentrations achieved during normal maternal epidural anesthesia do not affect M or N platelet aggregation or beta-tg release.     

Inhibition of baboon platelet aggregation in vitro and in vivo by the garlic derivative,ajoene

Abstract: Background: The infusion of pig growth factor‐mobilized peripheral blood leukocytes (containing 1 to 2% progenitor cells) (pPBPC) into baboons is associated with a thrombotic microangiopathy, which results from a direct effect of these pig cells on platelet aggregation. Ajoene is a synthetic derivative of garlic that inhibits aggregation of human platelets induced by all known agents. To assess its potential use in models of xenotransplantation, this agent was tested for its effect on baboon platelet aggregation in vitro and in vivo. Methods: In vitro studies: Baboon platelet aggregation assays, using adenosine diphosphate (ADP) (20 or 40 μm ) or collagen (12.5 μg/ml), were performed after incubation with ajoene (0 to 150 μg/ml) or dipyridamole (0 to 200 μg/ml). Platelets were also incubated with pPBPC (5 × 10⁶ cells) without or with ajoene in the absence of a known agonist. In vivo studies: Baboons received either a single intravenous dose of ajoene (10 to 25 mg/kg) or dipyridamole (0.8 mg/kg), or repeated doses of both agents at 2 to 3 h intervals. Platelet‐rich plasma was obtained for platelet aggregation assays at time points up to 4 h post‐drug administration. Results: In vitro, platelet aggregation was inhibited by 95% (ADP assay) and 89% (collagen assay) by ajoene at concentrations of ≥75 μg/ml. Dipyridamole had no effect at concentrations of <100 μg/ml, but inhibited aggregation almost completely at higher concentrations. Ajoene inhibited the aggregation caused by pPBPC by 33 to 50%. In vivo, platelet aggregation was completely inhibited for 2 h by ajoene at 25 mg/kg. Dipyridamole at 0.8 mg/kg reduced aggregation by 20% for 15 min, but the effect was lost by 60 min. In combination, the two agents prolonged inhibition marginally. Repeated doses of both agents at 2 h intervals maintained complete inhibition of aggregation, but did not do so when the interval between doses was extended to 2.5 or 3 h. Combined therapy was not associated with any bleeding complications. Conclusions: Although ajoene is a powerful inhibitor of platelet aggregation, the need for repeated administration and its partial effect on pPBPC‐induced platelet aggregation would suggest that it may be of only limited value in preventing the thrombotic microangiopathy that develops when pPBPC are infused into baboons. However, it would seem worthy of further investigation when used in combination with other agents.     

Cyclosporin A augments human platelet sensitivity to aggregating agents by increasing fibrinogen receptor availability

Clinical use of cyclosporin A (CsA) has been associated with platelet hypersensitivity and an increased incidence of thrombotic and vasoactive events. The purpose of this study was (1) to confirm that CsA enhances platelet sensitivity to the soluble agonists, adenosine diphosphate (ADP) and epinephrine (EPI), and (2) to determine if this enhancement is mediated by alteration in the availability of platelet surface fibrinogen receptors, a final mediator of platelet activation. Mean log dose of ADP required to achieve complete second-wave platelet aggregation in vitro decreased from 1.90 to 1.49 microM (n = 19, paired t test, P less than 0.05) and 2.86 to 2.11 microM (n = 16, P less than 0.05) following a 15-min and 3-hr incubation in the absence (saline) and presence of CsA (1000 ng/ml), respectively. At the threshold dose of ADP, concurrent thromboxane B2 levels at 15 min were 245 +/- 44 ng/ml (n = 12, saline) and 265 +/- 54 ng/ml (n = 9, CsA; P greater than 0.05). At 3 hr respective levels were 333 +/- 57 and 442 +/- 81 ng/ml (P greater than 0.05). Similar results were obtained with EPI. The number of fibrinogen binding sites in response to 50 microM ADP was determined in washed platelets in the absence and presence of CsA by radioligand binding. In 6 of 7 volunteers, CsA increased fibrinogen receptors from 26,635 +/- 4841 to 35,925 +/- 7290 sites/platelet (means +/- SEM; P less than 0.05). No change in receptor affinity was noted. In conclusion, cyclosporine does augment platelet reactivity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of the sulphonylurea drugs gliclazide and glibenclamide on blood glucose control and platelet function.

Platelet aggregation and adhesion are commonly increased in diabetes mellitus. These abnormalities may in part be responsible for the increased incidence of vascular disease in diabetics. We have investigated the effects of diet, diet plus glibenclamide, and diet plus gliclazide on plasma glucose control and platelet function in 10 newly diagnosed maturity-onset diabetics who had not previously been treated. Before treatment, the mean postprandial plasma glucose value was 13,4 +/- 0,8 mmol/l, which fell insignificantly on dietary treatment, to 12,2 +/- 1,0 mmol/l (P greater than 0,05). Both glibenclamide and gliclazide, when added to the diet, significantly lowered mean plasma glucose values to 9,3 +/- 0,8 mmol/l and 7,8 +/- 0,8 mmol/l respectively (P less than 0,05). Platelet aggregation in response to 1 mumol adenosine diphosphate (ADP) was increased in the diet period, whereas aggregation in response to 10 mumol and 100 mumol was normal. This suggests an increased sensitivity of the platelets to ADP in diabetic patients. The addition of both glibenclamide and gliclazide reduced the magnitude of the response to within the normal range. Platelet aggregation in response to 10 mumol adrenaline and 750 micrograms/ml collagen was significantly reduced by glibenclamide (P less than 0,05). We conclude that sulphonylurea therapy appears to reduce the increased platelet aggregation which occurs in diabetics. This may play a role in the prevention of vascular disease.     

Effect of Oral and Intravenous Diltiazem on Whole Blood Platelet Aggregability

Diltiazem, a calcium channel blocker, is known to suppress platelet function in vitro. However, its in vivo effects on platelets have been controversial. The effect of diltiazem on both human and calf platelets was studied using impedance whole-blood ag-gregometry. In vitro, platelet aggregation induced by ADP (5 μM) or collagen (1 μg/ml) was significantly suppressed by 0.1 to 1.0 mM (45 to 450 μg/ml) of diltiazem in both species. In calves weighing 70 to 90 kg, platelet aggregability did not change after the oral administration of 180 mg diltiazem. In this condition, diltiazem was not detected in plasma samples. Intravenous injection of 0.5 mg/kg diltiazem also did not significantly alter the aggregability, although the PR interval on the ECG elongated (40 msec, p <. 05), mean arterial pressure dropped (14 mmHg, p <. 05), and diltiazem was readily detected in plasma (199 ng/ml, 0.4 μM). In conclusion, platelet aggregability is not altered by a clinical dosage of diltiazem that is sufficient to induce hemodynamic changes with therapeutic plasma concentrations.     

HALOTHANE STIMULATES THE AGGREGATION OF PLATELETS OF BOTH NORMAL INDIVIDUALS AND THOSE SUSCEPTIBLE TO MALIGNANT HYPERTHEMIA

Platelet responses to halothane in normal individuals and inpatients susceptible to malignant hypothermia were evaluated.Platelets in platelet-rich plasma from both normal controlsand patients underwent aggregation in response to halothane.There was no significant difference in the degree of aggregationbetween normal subjects and patients. Aggregation by halothanewas associated with a change in platelet shape, centralizationof platelet granules, and phosphorylation of platelet actinbinding protein, myosin light chain, and a 40 000-dalton protein.Aggregation induced by halothane could be inhibited by EGTA,PGE₁, adenosine and verapamil, but not by aspirin. Aggregationinduced by halothane could be potentiated by small doses ofadrenaline or ADP and in some individuals by caffeine. However,previous exposure of platelets to halothane made them subsequentlyless aggregable to ADP. The results of these studies do notsupport a use of halothane-induced aggregation of plateletsto detect an abnormality in individuals susceptible to malignanthyperthermia, but do provide new evidence of the effects ofhalothane on cellular function.     

Soluble leptin receptor in serum of subjects with complete resistance to leptin: relation to fat mass

Leptin resistance and obesity have been related to mutations of the leptin receptor gene in rodents and, recently, in a consanguineous family. The latter mutation results in a receptor lacking transmembrane and intracellular domains. Homozygous and heterozygous individuals with this mutation had serum leptin levels higher than expected, given their BMIs: 600, 670, and 526 ng/ml and 145, 362, 294, 240, and 212 ng/ml, respectively. Their serum leptin was fractionated by gel filtration: >80% was present as a high-molecular size complex vs. 7.5% in the nonmutated sister. Western blot analysis showed a band at 146 kDa reacting specifically with an antibody directed against the leptin receptor ectodomain. In 10 obese control subjects, as in the mutated patients, free leptin levels correlated with BMI (r = 0.70, P = 0.0011) and reflected fat mass, regardless of leptin receptor functioning. In the patients, bound leptin levels correlated with BMI (r = 0.99, P = 0.0002) and were related to the number of mutated alleles. These data demonstrate that the truncated receptor is secreted into blood and binds the majority of serum leptin, markedly increasing bound and total leptin. Free serum leptin was similarly correlated with BMI in the mutated and nonmutated obese individuals, providing evidence that the relationship between BMI and circulating free leptin is preserved in this family. This finding suggests that the leptin receptor itself may not be specifically involved in the control of leptin secretion, and it supports the concept of relative resistance to leptin in common obesity.     

Insulin Resistance,Leptin and TNF-α System in Morbidly Obese Women after Gastric Bypass

Obesity is a complex disease associated with insulin resistance. Leptin and the TNF-α system could be involved in the pathogenesis of obesity and insulin resistance. Gastric bypass (GBP) is a surgical treatment for morbidly obese patients. We conducted a study after GBP to analyze the pattern of variation of anthropometric and body composition variables, leptin and sTNFR1 and 2. Methods: 29 morbidly obese women were studied, at baseline and throughout 6 months after gastric bypass. Results: At baseline, the BMI was 49 ± 6 kg/m² and patients showed a higher fasting insulin resistance index (FIRI), leptin, leptin/fat mass and sTNFR1 and 2 than did controls. 6 months after GBP, BMI was 35±4, and FIRI, leptin and leptin/fat mass decreased significantly in the first months and throughout the follow-up. sTNFR1 and 2 showed an initial increase, but at 6 months their concentrations were similar to baseline (2.6±0.8 vs 3.1±0.95 ng/ml, P < 0.05; 4.6±1.4 vs 7±2.5 ng/ml, P < 0.05). At baseline, there was no correlation between leptin and BMI and body composition variables but there was a correlation with fat mass (r=0.42, P=0.004) and sTNFR1 (r=0.58, P=0.001). At 6 months, there was a correlation between leptin and BMI (r=0.53, P=0.004) and sTNFR1 (r=0.46, P=0.013). Conclusions: Morbidly obese women after GBP became less insulin resistant with lower leptin concentrations, but showed an initial increase of sTNFR1 and 2. This pattern of variation of the leptin TNF-α axis suggests a disregulation of the system after dramatic weight loss and also that insulin and leptin up-regulate TNF-α production irrespective of insulin resistance status.     

Platelet function and anesthetics in cardiac surgery: an in vitro and ex vivo study.

We studied the effects of the anesthetics commonly used in cardiac surgery on platelet function. Fentanyl, droperidol, succinylcholine, pancuronium, thiopental, and diazepam at therapeutic concentrations were tested for their in vitro effects on the expression of platelet membrane glycoproteins Ib and IIbIIIa (GpIb, GpIIb-IIIa) and of P-selectin in anticoagulated whole blood by flow cytometry. The expression of P-selectin was determined under basal conditions, after the incubation of blood with adenosine diphosphate (ADP) 10 micromol/L, and the stable prostaglandin endoperoxide analog U46619 1 micromol/L. No drug affected the expression of P-selectin in unstimulated and ADP- or U46619-stimulated platelets, with the exception of thiopental, which markedly decreased the U46619-induced expression of P-selectin. Thiopental concentration-dependently inhibited U46619-induced and ADP-induced platelet aggregation, with effects on U46619-induced aggregation at therapeutic concentrations. To assess ex vivo effects, the same platelet markers were also assessed in blood obtained from 10 patients undergoing elective coronary surgery. Compared with basal values, platelet response to U46619 was significantly reduced just after the administration of anesthetic drugs, and the effect persisted for 48 h after surgery. Our study suggests that, at therapeutic concentrations, thiopental inhibits U46619-induced platelet activation both in vitro and ex vivo. The mechanisms responsible of this effect, together with its clinical significance, require further investigation. IMPLICATIONS: Thiopental inhibited prostaglandin-induced platelet activation at therapeutic concentrations both in vitro and ex vivo in cardiac surgical patients whereas adenosine diphosphate-induced activation was affected only at supratherapeutic drug concentrations. Thus, administration of sodium thiopental may contribute to the in vivo impairment of platelet function in patients undergoing elective cardiac surgery.     

Platelet size and function in septic rats: changes in the adenylate pool

Cecal ligation and puncture (CLP) were performed in rats. After 4 hr (early sepsis) and 16 hr (late sepsis), platelet morphology and function were studied. At 16 hr, platelet counts for the CLP group were significantly lower than for the sham-operated control group. Low maximum aggregation rates (MAR) and decreased platelet counts were elicited in platelet-rich plasma with 4 M ADP and 2 micrograms/ml collagen. However, with platelet counts equalized, MAR for the CLP group increased significantly, especially after 16 hr. The platelet-large cell rate and platelet distribution width decreased temporarily at 4 hr, then rose significantly at 16 hr. No significant changes were observed in the mean platelet volume after 4 hr, but there were significant increases after 16 hr. Total adenine nucleotide (TAN) levels within the platelets rose significantly in the CLP group, suggesting the appearance during the late sepsis of large, heavy platelets or adenine nucleotide-rich platelets. The platelet adenylate pool was divided into granular and cytoplasmic fractions, respectively characterized by ADP and ATP increases. However, no septicemia-related differences were noted in the degree of binding between goat antirat fibrinogen and platelet surface glycoprotein IIb/IIIa complex. Internal environment changes in the platelets indicated that during septicemia hyperfunctional or hypersensitive platelets with a latent capacity for active aggregation and release appeared in the circulation. Hypercoagulability in septicemia involves activation of coagulation factors, stimulation of the coagulation cascade, volume changes accompanying increased platelet TAN content, and changes in AN distribution in the two pools. These findings significantly increase our understanding of the transition from the prethrombotic state to thrombosis in septicemia.     

Effect of local hemostatics on platelet aggregation

The platelets play an important role in the normal hemostasis, and it is known that both natural and synthetic macromolecules may induce platelet activation and aggregation. Thus, the purpose of the present study was to investigate the platelet aggregating effect of five different local hemostatics. Platelet aggregation was assessed by aggregometry. Unwoven fleece of bovine collagen polymer in fibrillar form induced aggregation in combination with small amounts of platelet agonists; ADP and adrenaline. Ordinary, nonabsorbable bone wax also induced aggregation in combination with the agonists, but larger concentrations of agonists were needed. Bioerodible polyorthoester with physical properties such as bone wax, oxidized cellulose and gelatin sponge did not promote platelet aggregation.     

The hemostatic mechanism after open-heart surgery. II. Frequency of abnormal platelet functions during and after extracorporeal circulation.

In a prospective study of 13 patients undergoing open-heart surgery with extracorporeal circulation, marked qualitative platelet function defects were observed in addition to the usually occurring drop of the thrombocyte count. At the end of bypass, the following test results were significantly abnormal: concentration of fibrinogen and of circulating fibrin degradation products, platelet count, platelet adhesiveness to glass beads, and platelet aggregation induced by low and high doses of ADP. One to 2 hours after neutralization of heparin with protamine sulfate all abnormal test results improved, but the template bleeding time was markedly prolonged in 10 patients. There was no correlation between length of bypass and platelet fall and between concentration of circulating fibrin degradation products and extent of platelet dysfunction. An apparent correlation was found between the length of the postoperative bleeding time and the number of units of blood transfued during surgery. The results of this study suggest that dilution of the patient's own platelets by nonviable platelets contained in 3-day-old transfused ACD blood and the production of a refractory state of the patient's circulating platelets to ADP induced aggregation played a significant role in the development of platelet function abnormalities during extracorporeal circulation.     

Alterations of adenine nucleotide metabolism and function of blood platelets in patients with diabetes

Increased activity of blood platelets contributes to vascular complications in patients with diabetes. The aim of this work was to investigate whether persisting hyperglycemia in diabetic patients generates excessive accumulation of ATP/ADP, which may underlie platelet hyperactivity. Platelet ATP and ADP levels, thiobarbituric acid-reactive species synthesis, and aggregation of platelets from patients with diabetes were 18-82% higher than in platelets from healthy participants. In patients with diabetes, platelet stimulation with thrombin caused about two times greater release of ATP and ADP than in the healthy group while decreasing intraplatelet nucleotide content to similar levels in both groups. This indicates that the increased content of adenylate nucleotides in the releasable pool in the platelets of diabetic patients does not affect their level in metabolic cytoplasmic/mitochondrial compartments. Significant correlations between platelet ATP levels and plasma fructosamine, as well as between platelet ATP/ADP and platelet activities, have been found in diabetic patients. In conclusion, chronic hyperglycemia-evoked elevations of ATP/ADP levels and release from blood platelets of patients with diabetes may be important factors underlying platelet hyperactivity in the course of the disease.     

多囊卵巢综合征患者卵巢黄素化颗粒细胞瘦素信号转导分子STAT3磷酸化表达的研究

目的检测多囊卵巢综合征(PCOS)患者卵巢黄素化颗粒细胞瘦素信号转导子和活化子STAT3磷酸化(p-STAT3)水平,并观察瘦素体外对培养卵巢黄素化颗粒细胞p-STAT3水平的影响,探讨瘦素在PCOS发病机制中的作用。方法选择行体外受精-胚胎移植(IVF-ET)治疗的肥胖型PCOS患者(肥胖PCOS组)、非肥胖型PCOS患者(非肥胖PCOS组)、排卵功能正常和单纯输卵管因素不育的肥胖(肥胖对照组)及正常体重妇女(正常对照组)各15例。采用免疫印迹技术检测卵巢黄素化颗粒细胞p-STAT3水平。同时将正常人卵巢黄素化颗粒细胞行体外培养,分别加入不同浓度的瘦素(0、10、100、1,000 ng/ml)培养48 h,观察瘦素体外对人卵巢黄素化颗粒细胞p-STAT3水平的影响。结果(1)肥胖型PCOS组、肥胖对照组、非肥胖PCOS组和正常对照组卵巢黄素化颗粒细胞p-STAT3水平分别为(24.28±0.51)、(21.31±1.32)、(11.69±0.67)、(9.03±0.20),实验组间比较有显著性差异(P<0.05),其中肥胖型PCOS组p-STAT3表达水平最高,其次依次为肥胖对照组、非肥胖PCOS组和正常对照组。各组之间两两比较,均有显著性差异(P<0.05)。(2)加入瘦素培养48 h后,卵巢黄素化颗粒细胞p-STAT3水平随瘦素浓度升高而增高,呈浓度依赖性,至瘦素浓度达到100 ng/ml时,p-STAT3水平达到高峰,随后呈下降趋势。结论瘦素通过介导JAK2/STAT3信号途径可能参与了肥胖型PCOS的发病机制。     

Patients' thromboembolic potential after carotid endarterectomy is related to the platelets' sensitivity to adenosine diphosphate

Background and purpose Postoperative microemboli in patients undergoing carotid endarterectomy are a significant risk factor for stroke. These emboli can be detected by intraoperative transcranial Doppler monitoring. They are not linked to technical error and are variable between patients. As it is known that platelets play a key role in arterial thrombosis, it was hypothesized that a patient's risk of postoperative carotid thrombosis was linked to the individual's platelet response to physiologic agonists. METHODS: Blood samples from 120 patients undergoing carotid endarterectomy were analyzed before surgery. Platelet aggregation was measured in response to adenosine diphosphate (ADP) (0.5 to 4 micromol/L), collagen (10 to 50 mg/mL), and arachidonic acid (3 or 6 micromol/L), and fibrinogen binding to GPIIb-IIIa was measured by whole blood flow cytometry in response to ADP (0.1 to 10 micromol/L) and thrombin (0.02 to 0.16 micro/mL). Patients underwent intraoperative transcranial Doppler monitoring for 3 hours after surgery, and platelet functional data of those who had >25 emboli in this period (n = 22) were compared with the data of those with <25 emboli (n = 88). RESULTS: The platelet response to ADP was significantly higher in the patients with >25 emboli, as measured both by aggregometry (P =.0012) and by flow cytometry (P <.0001). Platelet aggregation with collagen was also significantly higher in this group (P =.0018), but the response to thrombin was not statistically different in the two groups. In addition, there was no difference in the response to arachidonic acid between the groups. CONCLUSION: The platelet response to ADP may be linked to clinical outcome, and thus, specific ADP receptor inhibitors may be appropriate for this group of patients.