A shear-induced in vitro platelet function test can assess clinically relevant anti-thrombotic effects

Morphological features of haemostatic plugs formed in vitro under high shear forces were investigated. Electron microscopy confirmed the relevance of such haemostatic plug to a platelet-rich arterial thrombus, which is formed in vivo . In rat blood samples, the effects of anticoagulants and various antiplatelet agents on platelet reactivity (rate of haemostatic plug formation) and subsequent coagulation of the flowing blood were investigated. Haemostasis did not occur in citrated blood, and heparin greatly inhibited the shear-induced platelet reaction. Aspirin (1 mM), a thromboxane A(2) receptor antagonist (5 microM), a stable prostacyclin (0.55 nM), a stable prostaglandin E(1) (141 nM) and a phosphodiesterase inhibitor (100 microM) were tested. All these agents exerted significant inhibitory effect on shear-induced platelet reaction, including the inhibition of the very first phase of platelet plug formation, due to aggregation of shear-activated platelets. Except for the phosphodiesterase inhibitor, which prolonged clotting time, none of the above agents affected dynamic coagulation. These results suggest that the employed in vitro shear-induced thrombosis/haemostasis test can reveal in vivo the antithrombotic effect of various agents independently of their mechanism of action.     

Mild and moderate hypothermia increases platelet aggregation induced by various agonists: a whole blood in vitro study

The mechanisms causing temperature-dependent bleeding, especially in hypothermic patients, warrant clarification. Therefore the aim of this study was to investigate platelet aggregation at the clinically important temperature range of 30–34°C. After obtaining informed consent citrated whole blood was drawn from 12 healthy adult male volunteers, who had not taken any medication in the previous 14 days. After venipuncture blood samples were incubated at 37°C until platelet testing. Platelet aggregation was performed in whole blood using the impedance aggregometer Multiplate® at five different test temperatures between 30°C and 34°C. Aggregation responses at 37°C served as controls. At temperatures of mild and moderate hypothermia (30–34°C), overall platelet aggregation was increased compared to 37°C. Increases were recorded in response to collagen, thrombin receptor activating peptide and ristocetin between 31°C and 34°C and in response to adenosine diphosphate between 30°C and 34°C. Overall platelet aggregation is increased at mild and moderate hypothermia down to 30°C. These results indicate that bleeding complications reported in mildly hypothermic patients are not due to hypothermia-induced platelet inhibition. The pathomechanism of the overall increased platelet aggregation between 30°C and 34°C requires further detailed study.     

The effects of storage on platelet function in different blood products

Objectives: Reduced platelet (PLT) function during storage has been shown for buffy-coat-derived platelet concentrates (BCP) and apheresis platelet units (AP), while for whole blood (WB) it has not been well studied. The aim of this study was to investigate PLT function in these blood products throughout storage using a novel flow cytometric assay.

Methods: Flow cytometric measurement of agonist-induced platelet aggregation, CD62P expression and PAC-1 binding during storage in BCP, AP (1–9 days at 20°C) and WB (1–21 days at 2–6°C).

Results: PLT-aggregation capacity decreased from day 1 to day 7 for almost all product–agonist combinations (P?=?.004 to P?=?.029) with aggregation capacity of WB being similar to that of AP and BCP. WB aggregation capacity remained relatively unchanged from day 7 to day 21. For all blood products, the fraction of agonist-induced CD62P-expression remained high and the fraction of PAC-1 binding decreased during storage. WB PLTs underwent only small changes in CD62P expression and PAC-1 binding from day 7 to day 21.

Conclusion: This study found PLT aggregation in WB stored at 4°C to be as least as good as for BCP and AP stored at 20°C. WB retained significant PLT-aggregation capacity to day 21.     

In vitro evaluation of platelet concentrates suspended in additive solution and treated for pathogen reduction: effects of clumping formation

BackgroundPlatelet concentrates may demonstrate visual, macroscopic clumps immediately after collection following aphaeresis or production from whole blood, independently of the preparation method or equipment used. The relationship between the occurrence of clumping and their effect on in vitro quality of platelets was investigated.ResultsNo significant differences were found throughout storage between the groups. The lactate dehydrogenase levels increased in both groups; this increase was higher in the test group on the last day of testing, without there being a significant difference on day 2. In contrast, pH values on day 2 were significantly different between the test and control groups. Platelet-derived cytokines increased comparably during storage.DiscussionThe results confirm good in vitro quality and storage stability of platelets suspended in SSP+ and treated with the Intercept pathogen reduction system. The presence of “non-compacted” clumps in platelet concentrates does not appear to affect the in vitro quality of the platelets.     

Blockade of the human platelet GPIIb/IIIa receptor by a murine monoclonal antibody Fab fragment (7E3): Potent dose-dependent inhibition of platelet function

Summary The platelet glycoprotein (GP) IIb/IIIa receptor can bind fibrinogen, von Willebrand factor, and other adhesive ligands; this binding is the final common pathway mediating platelet aggregation. The purpose of this study was to evaluate the safety and platelet inhibitory characteristics of the Fab fragment of the murine monoclonal anti-GPIIb/IIIa 7E3 antibody (m7E3 Fab) when administered intravenously as a single bolus dose, as a single and repeat bolus dose, and as a single bolus dose followed by continuous infusions of varying duration. Various dosage regimens of m7E3 Fab were studied in 74 patients with stable angina. Dosage regimens included single doses of m7E3 Fab from 0.1 to 0.3 mg/kg, a single dose of 0.20–0.30 mg/kg, and a repeat dose of 0.05 mg/kg, or a loading dose followed by a continuous infusion of m7E3 Fab for up to 36 hours. To assess the effect of m7E3 Fab on platelet function, quantitative blockade of GPIIb/IIIa receptors, inhibition of ex vivo platelet aggregation, and template bleeding time were measured in all patients. Dose-dependent inhibition of platelet function was evident in response to escalating bolus doses of m7E3 Fab, with maximum inhibition observed at 0.25–0.30 mg/kg body weight; at the 0.30 mg/kg dose, mean (±SE) GPIIb/IIIa receptor blockade was 81±3%, ex vivo platelet aggregation in response to 20 µM ADP was 14±6% of baseline, and the median bleeding time was >20 minutes. Although platelet function gradually recovered following a single bolus injection, platelet inhibition could be sustained by continuous, low-dose infusion of the antibody. Platelet inhibition occurred within minutes, but m7E3 Fab that did not bind to platelets cleared rapidly from circulation. Sixteen percent of the m7E3 Fab-injected subjects exhibited low titer, human anti-murine antibody responses. No significant bleeding or allergic reactions were observed in any patients. One of the 74 patients developed transient thrombocytopenia soon after receiving m7E3 Fab. These studies establish that m7E3 Fab can be administered safely at doses that cause profound inhibition of platelet function.     

Platelet aggregability and platelet volume in the postoperative course: problems in the platelet aggregation test derived from the measurement of platelet volume

Platelet count, aggregability and volume in the postoperative course of 20 patients were examined. Platelet count was decreased on the 1st postoperative d, and increased on the 7th and 14th d compared with the preoperative value. The maximal aggregation rate of platelets induced by ADP was decreased on the 3rd postoperative d, and then recovered to the preoperative level. In contrast, platelet volume was only slightly increased on the 3rd postoperative d. In this study, there was no correlation between platelet aggregability and platelet volume in PRP. We have proposed one parameter, 'platelet concentration ratio' (platelet concentration in PRP/platelet concentration in whole blood). In the postoperative course, this concentration ratio changed depending on platelet volume, and possibly on other conditions of blood such as hematocrit, viscosity and specific gravity. The concentration ratio influenced the subpopulations of platelets in PRP. Platelet aggregation tests may be performed using PRP in which platelet subpopulations differ from those in whole blood, especially in the postoperative state.     

Use of ICHOR-platelet works to assess platelet function in patients treated with GP IIb/IIIa inhibitors.

Platelet glycoprotein GP IIb/IIIa inhibitors have been recently approved for use in treating patients with acute coronary syndromes and those undergoing PCI. The purpose of this study was to assess the feasibility of using a new device, the ICHOR platelet works, to detect platelet inhibition in patients undergoing PCI and treated with abciximab or tirofiban. The study was conducted at Baylor College of Medicine, Houston, Texas. Thirty patients undergoing PCI and treated with abciximab (n = 10) or tirofiban (n = 20) are included. Blood samples were obtained before, at 30 min, at 4 hr, and at 12 hr after starting the GP IIb/IIIa inhibitors and 2 hr after discontinuation. Baseline studies revealed > 95% platelet aggregability in all patients after exposure to ADP (20 microM). After starting tirofiban, 82%, 83%, and 82% of platelets were inhibited at 30 min, 4 hr, and 12 hr. Platelet inhibition decreased to 43% 2 hr after discontinuation of tirofiban. Similarly, ICHOR platelet works detected 91%, 92%, and 85% platelet inhibition at 30 min, 4 hr, and 12 hr after starting abciximab, respectively. Platelet inhibition decreased to 73% 2 hr after discontinuation. The ICHOR platelet works is a promising, simple, and rapid bedside method that may have clinical utility in assessing platelet inhibition in patients treated with GP IIb/IIIa inhibitors. Cathet Cardiovasc Intervent 2001;53:346-351.     

辛伐他汀可增强阿司匹林对胶原诱导的血小板活化的抑制作用

【摘要】 目的 探讨他汀类药物联合阿司匹林体外孵育对胶原诱导的血小板活化的影响。方法 采集河北医科大学第二医院门诊健康成人志愿者外周静脉血,制备血小板悬液,应用全血阻抗法、流式细胞术、Elisa等方法测定不同浓度辛伐他汀或普伐他汀单独或联合阿司匹林共同孵育对血小板聚集及活化的影响。 结果 辛伐他汀体外孵育可降低胶原诱导的血小板聚集率,联合阿司匹林,可进一步降低胶原诱导的血小板活化标志物表达及血栓素TXB2合成。结论 辛伐他汀体外孵育可抑制胶原诱导的血小板活化,与阿司匹林联合,可进一步增强后者对血小板聚集的抑制作用。     

A new platelet parameter,the mean platelet component,can demonstrate abnormal platelet function and structure in myelodysplasia

Summary The mean platelet component (MPC) is a new platelet parameter generated by the Bayer ADVIA^TM 120 full blood count analyser as part of the routine complete blood count (CBC) test cycle. We report a case of myelodysplasia with bleeding complications and abnormal template bleeding time in whom low mean platelet component parameters were associated with partial platelet granule deficiency, demonstrated by transmission electron microscopy. We suggest that the mean platelet component is an inexpensive and rapid test to screen for platelet dysfunction related to ultrastructural abnormalities in myelodysplasia.     

REVIEWLaboratory investigation of platelet function

Advancement in the understanding of the mechanisms of platelet activation and the development of new techniques for studying platelet function have created the challenge of a wide range of options for investigating patients with suspected platelet disorders and for studying the effects of anti-platelet drugs. Novel classes of anti-platelet drug, such as adenosine diphosphate (ADP) receptor and glycoprotein IIb/IIIa receptor antagonists, are being developed and introduced into routine clinical practice. This review presents an overview of current techniques for laboratory investigation of platelet function in the light of these recent advances.     

The impact of a medium molecular weight, low molar substitution hydroxyethyl starch dissolved in a physiologically balanced electrolyte solution on blood coagulation and platelet function in vitro

BACKGROUND AND OBJECTIVES: Hydroxyethyl starches (HES) may have the potential to impact negatively on haemostasis. Recent findings suggest that side-effects on haemostasis stem not only from the physicochemical differences between HES, but also from the composition of the solvent. We compared the effects of a newly developed medium molecular weight (MW) and low molar substitution (MS) HES dissolved in a physiologically balanced electrolyte solution (MW 130, MS 0.42; B-HES) with a commercially available non-balanced HES (MW 130, MS 0.4; NB-HES), and with Ringer's lactate (RL) solution in vitro. MATERIALS AND METHODS: Activated partial thromboplastin time (APTT), factor VIII clotting activity (F VIII:C) and von Willebrand factor (vWF) activity were investigated in 48 healthy individuals. Platelet function as measured by turbidimetric platelet aggregometry and whole blood impedance aggregometry induced by adenosine diphosphate (ADP), collagen and thrombin receptor activating peptide (TRAP), and by ADP and TRAP-induced expression of activated platelet fibrinogen receptor glycoprotein (GP) IIb/IIIa was determined in 24 participants. Haemodilution (25% and 50%, v/v for blood coagulation analyses and 20% and 40%, v/v for platelet function studies) was performed using the two HES preparations and RL. RESULTS: APTT was significantly longer and F VIII and vWF significantly lower at 25% and 50% dilutions with NB-HES compared to B-HES and RL. At 20% and 40% dilutions, ADP and TRAP-induced expression of activated platelet surface GP IIb/IIIa was significantly increased by B-HES compared to NB-HES and RL. Percentages of platelet GP IIb/IIIa expression were also significantly greater in samples diluted with B-HES than in undiluted blood. Neither the diluent (B-HES, NB-HES and RL) nor the degree of dilution (undiluted, 20% and 40% dilution) had any significant influence on ADP, collagen or TRAP-induced turbidimetric platelet aggregation or impedance platelet aggregation. CONCLUSIONS: In contrast to a non-balanced 130 kDa, MS 0.4 HES (NB-HES), a 130 kDa, MS 0.42 HES preparation dissolved in a physiologically balanced electrolyte solution (B-HES) does not affect APTT, F VIII:C and vWF in vitro. Both types of HES do not affect platelet aggregation induced by ADP, collagen or TRAP. B-HES but not NB-HES increases the expression of activated platelet GP IIb/IIIa induced by ADP or TRAP.     

Evaluation of platelet function in thrombocytopenia

Whole blood aggregometry is a functional assay for determination of platelet function. Until now, whole blood aggregometry has not been considered feasible at low platelet counts. Hence, the objectives of the present study were to explore platelet function in thrombocytopenia using a novel index of impedance aggregometry adjusted for platelet count and evaluate the association to platelet function assessed by flow cytometry. Hirudin anticoagulated blood was collected from 20 healthy volunteers, 20 patients with primary immune thrombocytopenia (ITP), and 17 hematological cancer patients. Platelet function was analyzed by impedance aggregometry and by flow cytometry. Collagen, adenosine diphosphate, thrombin receptor agonist peptide-6, and ristocetin were used as agonists for both analyses. Thrombocytopenia in healthy whole blood was induced in vitro employing a recently published method. Platelet aggregation of thrombocytopenic patients was evaluated relative to the aggregation of healthy volunteers at the same platelet count. In flow cytometry, platelet function was described as expression of the platelet surface glycoproteins: bound fibrinogen, CD63, and P-selectin. Similar platelet counts were obtained in the patient groups (p = 0.69) (range: 13–129 × 10⁹/l). Aggregation adjusted for platelet count was significantly increased in ITP patients compared to healthy platelets across all agonists. The platelet aggregation was high in the 95% prediction interval, with 18 ITP patients above the prediction interval in at least two agonists. In contrast, the platelet aggregation was low in the prediction interval in cancer patients, and three cancer patients with platelet aggregation below the prediction interval in at least one agonist. ITP patients displayed increased expression of bound fibrinogen and CD63 following activation, compared with particularly cancer patients, but also compared with healthy platelets. This study demonstrated the feasibility of a novel approach to perform platelet function analyses in thrombocytopenia using impedance aggregometry adjusted for platelet count.     

Platelets derived from fresh and cold‐stored whole blood participate in clot formation in rats with acute traumatic coagulopathy

The in vitro haemostatic functions of fresh whole blood (FWB) are well preserved after cold storage. This study aimed to determine whether platelets derived from FWB and stored whole blood (SWB) contribute to clot formation in tissue injury after transfusion into coagulopathic rats with polytrauma/haemorrhage (T/H). The rats were resuscitated 1 h after trauma with FWB or SWB collected from green fluorescence protein (GFP) transgenic rats. After transfusion, a liver incision was made and the tissue was collected 10 min after injury to identify GFP⁺ platelets by immunohistochemistry. In comparison to FWB, platelet aggregation to adenosine diphosphate and protease‐activated receptor‐4 was reduced by 35% and 20%, and clotting time was shortened by 25% in SWB. After transfusion, SWB led to a significant increase in platelet activation as measured by an elevation of CD62P and phosphatidylserine expression. The platelets from SWB were in a higher activation state, and showed higher clearance rate and formation of platelet‐leucocyte aggregates than those from FWB after transfusion. Platelets from both FWB and SWB were equivalently incorporated into the clot at the incisional site, as determined by co‐localization of CD61 and GFP. This study suggests that SWB contributes to haemostatic function and is an effective alternative resource to treat trauma patients.     

Bleeding time in uremia: a useful test to assess clinical bleeding

Modified Ivy bleeding time (template) and platelet aggregation to ADP, epinephrine, and collagen were studied in 26 uremic patients who had not recently ingested anti-platelet drugs. Regardless of the aggregating agent used, the abnormalities in platelet aggregation were often mild, even with advanced uremia, and frequently less severe than the effects of common anti-platelet drugs. The inhibition of collagen-induced aggregation was significantly correlated with both increased bleeding time and blood urea nitrogen. Platelet aggregation was not discriminative between clinically bleeding and non-bleeding groups of patients, but the bleeding time was helpful in this regard. In certain cases, the aggregometric patterns differed between drug-induced and uremic thrombocytopathies. Platelet aggregometry appears to be of little help clinically in assessing the severity of the uremic bleeding diathesis.     

Availability of endothelial von Willebrand factor and platelet function in diabetic patients infused with a vasopressin analogue

Summary Acute release of von Willebrand factor and its immunologically detected correspondent, factor VIII-related antigen, from the endothelial cells into the circulation was induced by an infusion of 1-deamino-8-D-arginine vasopressin in eight normal subjects and eight insulin-dependent diabetic patients. The patients had higher basal levels of von Willebrand factor (140±13%) and VIII-related antigen (112±18%) than the normal subjects (95±5% and 70±8%, respectively). 1-deamino-8-D-arginine vasopressin induced a two fold rise of these levels in both groups, von Willebrand factor/ VIII-related antigen remaining higher in the diabetic patients throughout the experiment. In the normal subjects, platelet retention in glass bead columns increased soon after the infusion but returned to basal values after 2 h. In the patients, it remained high at the end of the infusion. No changes in platelet aggregation occurred. These results suggest that increased plasma von Willebrand factor/VIII-related antigen in diabetic patients is accompanied by an increased endothelial content of this factor.     

Impairment of platelet function in both mild and severe COVID-19 patients

Abnormalities of platelet function were reported in patients with severe COVID-19 (severe-C), but few data are available in patients with mild COVID-19 (mild-C) and after COVID-19 recovery. The aim of this study was to investigate platelet parameters in mild-C patients (n = 51), with no evidence of pneumonia, and severe-C patients (n = 49), during the acute phase and after recovery, compared to 43 healthy controls. Both mild-C and severe-C patients displayed increased circulating activated platelets, low δ-granule content (ADP, serotonin), impaired platelet activation by collagen (light transmission aggregometry) and impaired platelet thrombus formation on collagen-coated surfaces under controlled flow conditions (300/s shear rate). The observed abnormalities were more marked in severe-C patients than in mild-C patients. Overall, 61% (30/49) of mild-C and 73% (33/45) of severe-C patients displayed at least one abnormal platelet parameter. In a subgroup of just 13 patients who showed no persisting signs/symptoms of COVID-19 and were re-evaluated at least 1 month after recovery, 11 of the 13 subjects exhibited normalization of platelet parameters. In conclusion, mild abnormalities of platelet parameters were present not only in severe-C but also, albeit to a lesser extent, in mild-C patients during the acute phase of COVID-19 and normalized in most tested patients after clinical recovery.     

Pharmacodynamic effects of clopidogrel in pediatric cardiac patients: A comparative study of platelet aggregation response

Little data on pediatric percent platelet aggregation (%PA) exist in the literature, particularly in cardiac patients and in response to clopidogrel. The objectives were to estimate the %PA range expected in pediatric patients and to measure the clopidogrel effect on %PA in the PICOLO (Platelet Inhibition in Children on Clopidogrel) trial. To estimate a neonatal/infant %PA response range, %PA induced by 5?µM adenosine diphosphate (ADP) was assessed using light transmission aggregometry in 16?cord and 11 normal adult blood samples and prior to clopidogrel therapy in 49 neonatal and 49 infant/toddler cardiac patients enrolled in PICOLO. The %PA induced by 5?µM thrombin receptor-activating peptide (TRAP) was also assessed for 10 neonates and 21 infants/toddlers enrolled in PICOLO and compared with 11 adult samples. Percent inhibition of platelet aggregation (%IPA) induced by 5?µM ADP at steady-state clopidogrel levels was assessed in 33 neonates and 39 infants/toddlers. ADP-induced %PA was lowest in cord blood samples, intermediate in study neonates and infants/toddlers, and highest in adults. Similarly, TRAP-induced platelet aggregation was lower in neonates and infants/toddlers than adults. For all groups, %PA and %IPA were highly variable, with 11% of neonates and 13% of infants/toddlers showing <10% IPA. In conclusion, ADP- and TRAP-induced %PA is lower in pediatric cardiac patients than normal adults, but highly variable in both. The lower baseline %PA may explain why the pediatric clopidogrel dose providing 30–50% IPA (0.20?mg/kg/day) is lower than a simple weight-based extrapolation of the adult dose (75?mg/day) providing similar inhibition.     

In vivo and in vitro effects of different anaesthetics on platelet function

Different effects of thiopental, propofol and sevoflurane on platelets have been reported. Patients undergoing thyroid surgery were anaesthetized with thiopental-fentanyl-sevoflurane (n = 11) or propofol-fentanyl-sevoflurane (n = 9). Platelet aggregation and thromboxane A2 generation were studied at baseline, and at the end of anaesthesia induction and surgery. Dose-response experiments were also performed in vitro with single agents. Thiopental-fentanyl-sevoflurane significantly reduced collagen-induced aggregation by the end of induction, while ADP-induced aggregation and thromboxane generation were unaffected. Propofol-fentanyl-sevoflurane had no effect on platelets. Thiopental dose-dependently inhibited platelets in vitro, while fentanyl or propofol did not. In conclusion, thiopental reduces platelet function both ex vivo and in vitro and propofol might be considered haemostatically safer.