Platelet function in platelet concentrates and in whole blood

Platelet function was studied in CPD whole blood stored at 4 degrees C for one and three days and in platelet concentrates stored at room temperature for the same periods of time. Comparisons were made of platelet shape, nucleotide content, beta-thromboglobulin (beta TG) liberated during storage, and platelet aggregation in response to ADP, collagen, sodium arachidonate and ristocetin. It was found that in whole blood the shape of the platelets was less discoid than in platelet concentrates. However, platelet aggregation in response to ADP, collagen, and sodium arachidonate was preserved better in whole blood than in platelet concentrates. Platelet nucleotides were the same in whole blood as in platelet concentrates, but the plasma levels of beta TG were less in whole blood. The results show that as judged by aggregation, beta TG release and nucleotide content, platelets from whole blood were at least as functional as those from platelet concentrates. However, platelets from whole blood had lost their discoid shape, which suggests that they would have a short survival in the circulation.     

Glycoprotein IIb/IIIa receptor inhibitor attenuates platelet aggregation induced by thromboxane A2 during in vitro nonpulsatile ventricular assist circulation

A recent development in antithrombotic research allows the inhibition of platelet aggregation via protection of the glycoprotein IIb/IIIa receptor on the platelet membrane. We hypothesized that a GP IIb/IIIa receptor inhibitor would inhibit thromboxane-induced platelet aggregation during circulation in our in vitro ventricular assist device (VAD) circuit and preserve long-term platelet function. Twenty-one in vitro nonpulsatile centrifugal VAD circuits were simulated for 4 days using 450 ml of fresh human whole blood with or without glycoprotein IIb/IIIa receptor inhibitor (tirofiban). Platelet aggregation and degranulation were measured in whole blood induced by ristocetin, collagen, ADP, and thromboxane A2 (TXA2). The tirofiban-treated group preserved the platelet count and tended to exert these beneficial effects by inhibiting pathologic platelet aggregation induced by TXA2, collagen, and ADP as well as degranulation. Tirofiban may be useful in preserving platelet number and function during clinical VAD use.     

Aprotinin-ACD-blood: I. Experimental studies on the effect of aprotinin on the plasmatic and thrombocytic coagulation (author's transl)

Aprotinin interrupts the spontaneous formation of microaggregates in stored blood. The analysis of the aprotinin-induced effects on the plasmatic and thrombocytic clotting systems showed a correlation between concentration and inhibitory effects of aprotinin on clotting factor VIII, IX, XI, XII as well as on the enzyme-induced second phase aggregation of the platelets. The effect of aprotinin on inhibition of platelet aggregation is probably due to binding of enzymes located in the membrane. The membrane stabilizing effect of aprotinin is discussed. Also in different experimental models using enzyme kinetic designs under maximal aprotinin inhibition a normalization of platelet functions could be immediately reached under transfusion analogous conditions. It is therefore, assumed that transfusion of aprotinin-ACD-Blood does not alter the coagulation mechanism of the patient.     

The effect of hypotensive anesthesia with nitroglycerin and verapamil on platelets

Recently, there have been many cases in which nitroglycerin (TNG) is used for anesthesia for patients with low blood pressure. Since, it has been reported that TNG suppresses the platelet aggregation function in vitro. Verapamil (Vp), known as another platelet aggregation suppressant, and nicardipine (Nc) were used together with TNG and the platelet aggregation function was studied in vitro and in vivo. The results of the in vitro experiment showed that TNG 250 ng.ml-1 + Vp 250 ng.ml-1 suppressed the platelet aggregation function, and when TNG + Vp (was used for low hypotensive anesthesia, a large amount of Vp i.e. over 15 mg of Vp is injected as a bolus into the artery, there is a possibility the platelet aggregation function will be suppressed. No platelet aggregation function suppression was found when TNG+Nc was used in hypotensive anesthesia.     

Platelet aggregation during and after general anesthesia and surgery

Because of evidence suggesting that volatile anesthetics interfere with platelet aggregation in vitro, platelet function was investigated in patients exposed to combinations of nitrous oxide, halothane, and enflurane during brief surgical procedures. In 12 patients undergoing elective operations, blood was sampled before anesthesia, after induction but prior to surgery, during surgery, and postoperatively. Platelet aggregation by ADP, epinephrine, and collagen was similar during each sampling period in all but two subjects in whom only minor changes were noted. During brief exposure to these general anesthetics, there appears to be no clinically important inhibition of platelet function.     

4%琥珀酰明胶、6%羟乙基淀粉和12%缩合葡萄糖对血小板聚集功能的体外研究

目的研究4％琥珀酰明胶(佳乐施，GF)、6％羟乙基淀粉(贺斯，HES)和12％缩合葡萄糖(益极疏，PG)三种胶体液对血小板聚集功能的影响。方法采健康人静脉血(n=10)，分为对照组(NS组)、GF组、HES组和PG组。以全血值为基础值，每组分为8：2和6：4两个稀释度。以二磷酸腺苷(ADP)为诱导剂．观察各组血小板最大聚集率(PAg Tmax)和聚集坡度(Slope)的变化(涡流密度测量法)。结果排除稀释因素影响，各胶体组均不同程度地抑制血小板聚集，PG的抑制作用最强，其次是HES，GF作用最小。结论胶体液抑制血小板聚集的作用是其抑制凝血功能的重要组成部分，临床应根据病人凝血功能状态合理选择不同的胶体液，以策安全。     

The effects of bupivacaine and pipecoloxylidide on platelet function in vitro

The influence of bupivacaine and its major metabolite, pipecoloxylidide, on human platelet function was studied in vitro. Significant inhibition of ADP and collagen-induced platelet aggregation occurred only with concentrations of bupivacaine above 10 micrograms.ml-1. This concentration (10-25 micrograms.ml-1) is much higher than would be expected in routine clinical use of bupivacaine for epidural analgesia. The inhibition of platelet aggregation was associated with a significant decrease in beta-thromboglobulin secretion. In contrast, pipecoloxylidide had no effect on platelet aggregation or the beta-thromboglobulin release. We conclude that the previously reported 30-min time-lag between the maximal plasma concentration of bupivacaine and the inhibition of platelet aggregation is unlikely to be due to a metabolism of bupivacaine to pipecoloxylidide.     

The effect of anaesthetic agents on platelet function.

This paper reviews studies which have investigated the effect of anaesthetic agents on platelet function. The results of these studies suggest that halothane is the only agent in current use which inhibits platelet function in concentrations used clinically. Nitrous oxide appears to cause only a modest inhibition, while enflurane and isoflurane appear to have minimal or negligible effects. There is no current evidence that intravenous induction agents, opiates, or muscle relaxants affect platelet function. Reports indicate that local anaesthetic agents inhibit platelet aggregation, but only at concentrations far greater than peak plasma concentrations found during clinical use. Epidural anaesthesia may be associated with a reduction in platelet aggregation through a mechanism unrelated to direct local anaesthetic inhibition. The clinical significance of the effect of halothane on platelet function is not known. However, it is possible that halothane may affect bleeding or thrombotic complications in a similar manner to other 'anti-platelet' drugs.     

Functional changes in platelets during extracorporeal circulation.

An in vitro trauma test was conducted to determine the effects of extracorporeal circulation on platelet count and function. Fresh human blood was circulated in two identical in vitro circuits for six hours at a rate of 500 ml per minute (500 recirculations). One circuit included a G.E.--Peirce membrane lung and the other was a control. Platelet aggregation induced by adenosine diphosphate (ADP), epinephrine, or collagen was studied before and after six hours of perfusion. No important drop in platelet count occurred in the control circuit (Control-C) following bypass, but there was a 20% drop for the lung circuit (Lung-C). Platelet aggregation was reduced by about 30% for the control circuit and 65% for the lung circuit. The large decrease in platelet function accompanied by only a moderate decrease in platelet count is discussed in terms of loss of the youngest and most active platelets, platelet inhibition due to ADP released by red blood cell lysis, and platelet trauma.     

The effects of ropivacaine hydrochloride on platelet function: an assessment using the platelet function analyser (PFA-100)

Amide local anaesthetics impair blood clotting in a concentration-dependent manner by inhibition of platelet function and enhanced fibrinolysis. We hypothesised that the presence of ropivacaine in the epidural space could decrease the efficacy of an epidural blood patch, as this technique requires that the injected blood can clot in order to be effective. Ropivacaine is an aminoamide local anaesthetic used increasingly for epidural analgesia during labour. The concentration of local anaesthetic in blood achieved in the epidural space during the performance of an epidural blood patch is likely to be the greatest which occurs (intentionally) in any clinical setting. This study was undertaken to investigate whether concentrations of ropivacaine in blood, which could occur: (i) clinically in the epidural space and (ii) in plasma during an epidural infusion of ropivacaine, alter platelet function. A platelet function analyser (Dade PFA-100, Miami) was employed to assess the effects of ropivacaine-treated blood on platelet function. The greater concentrations of ropivacaine studied (3.75 and 1.88 mg x ml(-1)), which correspond to those which could occur in the epidural space, produced significant inhibition of platelet aggregation. We conclude that the presence of ropivacaine in the epidural space may decrease the efficacy of an early or prophylactic epidural blood patch.     

Pharmacological effect of nitroprusside on platelet aggregation

Adequate platelet function and numbers are critical for postcardiopulmonary bypass patients. Endogenous and pharmacological sources of nitric oxide (NO) are known inhibitors of platelet aggregation. Sodium nitroprusside (SNP), used clinically to control blood pressure, is an inorganic source of NO. Our long-term goal is to determine if SNP infusion in the venous return line of the cardiopulmonary bypass system would preserve platelet numbers and function without affecting systemic vascular resistance. Our first requirement to accomplish this goal was to develop an assay that would detect the SNP effect on platelet aggregation. We, therefore, tested the hypothesis that clinical concentrations of SNP would inhibit platelet aggregation. We quantified platelet aggregation with the Medtronic Hepcon HMS whole blood aggregometer. Normal heparinized human blood was treated with 0.625 to 12.5 nM platelet activating factor (PAF), 0.25 to 5.0 microM epinephrine, or 0.20 to 10 microM adenosine 5'-diphosphate (ADP) to stimulate platelet aggregation. SNP was added at 10(-5) M to determine its affect on PAF, epinephrine, and ADP stimulated platelet aggregation. The results demonstrated that PAF-stimulated platelet aggregation was significantly inhibited with SNP (10(-5) M) to 82% (p < .05) of control and epinephrine and ADP mediated aggregation were not significantly affected. In conclusion, at clinically relevant concentrations SNP inhibits platelet aggregation by PAF but not with ADP or epinephrine.     

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.     

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.     

Pump-induced platelet aggregation in albumin-coated extracorporeal systems.

OBJECTIVE: Coating of extracorporeal systems with heparin does not prevent platelet activation and subsequent bleeding disorders. We investigated whether this could be due to elevated shear stress caused by a roller pump. METHODS: Human or rat blood was made to flow through an uncoated or an albumin-coated medical polyvinyl chloride tube with or without a roller pump. Aggregation of platelets in the tubing was recorded continuously with a photometric device. RESULTS: Although in vitro gravitational flow in uncoated tubes caused immediate platelet aggregation and platelet loss, this remained absent in coated tubes. When the pump was started in experiments with a coated tube strong platelet aggregation was observed and platelet count fell within 5 minutes to 78% +/- 2% and 71% +/- 3% of control values in human and rat blood, respectively. In vivo, no aggregation was observed during spontaneous flow in rats with an albumin-coated tube running from the carotid artery to the femoral artery, but aggregation started as soon as the blood was pumped. Pump-induced platelet aggregation, both in vitro and in vivo, could be prevented with aurintricarboxylic acid, which specifically inhibits shear-induced platelet aggregation as has recently been shown. Pump perfusion of blood in an uncoated tube did not elicit platelet aggregation. CONCLUSIONS: Pump perfusion of blood in coated systems elicits shear-induced platelet aggregation, which may be prevented by administration of substances that block the binding of von Willebrand factor to glycoprotein Ib receptors on the platelets. The effects of pumping on platelets are masked in uncoated circuits because of the dominant influence of blood-material contact.     

Haematological effects of anaesthetics and anaesthesia

Halothane is still unique in its ability to inhibit platelet aggregation and to increase bleeding time in vivo at clinical concentrations, although sevoflurane inhibits platelet aggregation induced by weak agonists. Propofol itself, but not its fat emulsion, inhibits platelet aggregation and suppresses calcium mobilization. Extradural anaesthesia has been shown to prevent hypercoagulability during the perioperative period. Aprotinin reduces both blood loss and the incidence of blood transfusion during major orthopaedic and cardiac surgery.     

The effect of propofol on the interaction of platelets with leukocytes and erythrocytes in surgical patients

We tested the antiplatelet effect described for propofol in vitro in surgical patients. Platelet aggregation induced by adenosine diphosphate, collagen, and arachidonic acid was tested in samples of whole blood, platelet-rich plasma (PRP), PRP with red blood cells, and PRP with leukocytes. Also measured were platelet production of thromboxane (Tx)B(2) and leukocyte production of 6-keto-prostaglandin F(1 alpha) (a stable metabolite of prostacyclin) and plasma levels of nitrites + nitrates (indicator of nitric oxide production). Anesthesia was induced with a bolus IV injection of sodium thiopental 4 mg/kg (n = 10), with a bolus dose of 2.5 mg/kg of propofol (n = 20), or with propofol total IV anesthesia (n = 20). Sodium thiopental did not modify any of the analytical values. In patients who received a bolus injection of propofol, platelet aggregation was significantly reduced in whole blood and in PRP + leukocytes. Platelet production of TxB(2) was reduced by 35%; the inhibition of 6-keto-prostaglandin F(1 alpha) was not statistically significant. Plasma levels of nitrites + nitrates increased by 37%; this change correlated significantly with the decrease in systolic and diastolic blood pressure (both P < 0.05). Similar changes, albeit of larger magnitude, were seen in patients who were given total IV anesthesia with propofol. In conclusion, propofol inhibited platelet aggregation in surgical patients mainly as a result of the inhibition of Tx synthesis and the increase in nitric oxide production. These effects are thought to be related to the hypotensive effect of this anesthetic. IMPLICATIONS: In vitro experiments have shown that propofol inhibits platelet aggregation and increases nitric oxide production. This study shows that doses habitually used to induce or maintain anesthesia also have these effects. These findings have potential applications for patients at increased risk for bleeding and may partly explain the hypotensive effect of propofol.     

Effects of low temperature on shear-induced platelet aggregation and activation

BACKGROUND: Hemorrhage is a major complication of trauma and often becomes more severe in hypothermic patients. Although it has been known that platelets are activated in the cold, studies have been focused on platelet behavior at 4 degrees C, which is far below temperatures encountered in hypothermic trauma patients. In contrast, how platelets function at temperatures that are commonly found in hypothermic trauma patients (32-37 degrees C) remains largely unknown, especially when they are exposed to significant changes in fluid shear stress that could occur in trauma patients due to hemorrhage, vascular dilation/constriction, and fluid resuscitation. METHODS: Using a cone-plate viscometer, we have examined platelet activation and aggregation in response to a wide range of fluid shear stresses at 24, 32, 35, and 37 degrees C. RESULTS: We found that shear-induced platelet aggregation was significantly increased at 24, 32, and 35 degrees C as compared with 37 degrees C and the enhancement was observed in whole blood and platelet-rich plasma. In contrast to observation made at 4 degrees C, the increased shear-induced platelet aggregation at these temperatures was associated with minimal platelet activation as determined by the P-selectin expression on platelet surface. Blood viscosity was also increased at low temperature and the changes in viscosity correlated with levels of plasma total protein and fibrinogen. CONCLUSION: We found that platelets are hyper-reactive to fluid shear stress at temperatures of 24, 32, and 35 degrees C as compared with at 37 degrees C. The hyperreactivity results in heightened aggregation through a platelet-activation independent mechanism. The enhanced platelet aggregation parallels with increased whole blood viscosity at these temperatures, suggesting that enhanced mechanical cross-linking may be responsible for the enhanced platelet aggregation.     

Intermittent pneumatic foot compression can activate blood fibrinolysis without changes in blood coagulability and platelet activation

BACKGROUND: Intermittent pneumatic foot compression (IPC) is a useful technique for prophylaxis of peri-operative venous thromboembolism. The aim of this study was to determine the effect of IPC on blood coagulation/fibrinolysis and platelet function using a blood viscometer (Sonoclot) and a platelet aggregation monitor (WBA analyzer(TM)), respectively. Using the same blood samples, serum levels of tissue-type plasminogen activator (t-PA), thrombomodulin (TM) and activated protein C (APC) were also measured. METHODS: The soles and legs of each subject (n = 8) were compressed for 3 s (130 mmHg) at a 0.3-Hz interval using an IPC device. Parameters were measured 2 min before and at the end of 60-min compression. RESULTS: Parameters of the Sonoclot time-to-peak were shortened and clot retraction rate was increased significantly by IPC, whereas the other parameters did not change. These results indicate that IPC can activate blood fibrinolysis but not coagulability. A parameter of the WBA analyzer PATI (platelet aggregatory threshold index) did not change, indicating that IPC cannot activate platelet function per se. The concentration of t-PA decreased slightly but significantly. A decrease in the concentration of t-PA can lead to activation of fibrinolysis. Other humoral parameters did not change, indicating that IPC has no effect on endothelial function. Although neither blood coagulability nor platelet function were affected by IPC, fibrinolytic activity increased slightly, probably by activation of t-PA function. CONCLUSION: IPC is useful for prophylaxis for thromboembolism by activation of blood fibrinolysis as well as inhibition of blood stasis.     

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.     

The effect of ketanserin on blood pressure and platelets during cardiopulmonary bypass

Ketanserin, a serotonin antagonist, was used to control blood pressure during cardiopulmonary bypass in 12 patients having cardiac surgery. The drug was administered as a 10 mg bolus followed by a continuous infusion of either 40, 80, or 120 mg/hr to maintain mean arterial blood pressure below 70 mm Hg. There were 16 hypertensive episodes of which 15 (93.7%) were successfully controlled with ketanserin. Mean arterial pressure decreased significantly from an average of 72 +/- 3 to 52 +/- 9 mm Hg after 1 min. The effect that ketanserin had on platelets was also evaluated. Neither adverse nor salutary effects were seen in the platelet count, though a significant inhibition of serotonin-induced platelet aggregation was observed. Ketanserin proved effective for controlling hypertension during cardiopulmonary bypass but, despite inhibition of serotonin-induced platelet aggregation, it did not prevent thrombocytopenia.