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Objectives: Previous studies have described impaired platelet function after cardiopulmonary bypass (CPB). Whether this is still valid in contemporary cardiac surgery is unclear. This study aimed to quantify changes in function and number of platelets during CPB in a present-day cardiac surgery cohort., Design: Prospective, controlled clinical study., Setting: A single-center university hospital., Participants: Thirty-nine patients scheduled for coronary artery bypass graft surgery with CPB., Interventions: Platelet function and numbers were measured at 6 timepoints in 39 patients during and after coronary artery bypass graft surgery; at baseline before anesthesia, at the end of CPB, after protamine administration, at intensive care unit (ICU) arrival, 3 hours after ICU arrival, and on the morning after surgery., Measurements and Main Results: Platelet function was assessed with impedance aggregometry and flow cytometry. Platelet numbers are expressed as actual concentration and as numbers corrected for dilution using hemoglobin as a reference marker. There was no consistent impairment of platelet function during CPB with either impedance aggregometry or flow cytometry. After protamine administration, a decrease in platelet function was seen with impedance aggregometry and for some markers of activation with flow cytometry. Platelet function was restored 3 hours after arrival in the ICU. During CPB (85.0 ± 21 min), the number of circulating platelets corrected for dilution increased from 1.73 ± 0.42 × 10 9 /g to 1.91 ± 0.51 × 10 9 /g (p < 0.001)., Conclusions: During cardiac surgery with moderate CPB times, platelet function was not impaired, and no consumption of circulating platelets could be detected. Administration of protamine transiently affected platelet function., Competing Interests: Conflicts of Interest None., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Heparin and protamine are fundamental in the management of anticoagulation during cardiac surgery. Excess protamine has been associated with increased bleeding. Interaction between protamine and platelet function has been demonstrated but the mechanism remains unclear. We examined the effect of protamine on platelet function in vitro using impedance aggregometry, flow cytometry, and thrombin generation. Platelets were exposed to protamine at final concentrations of 0, 20, 40, and 80 µg/mL, alone or together with adenosine diphosphate (ADP) or thrombin PAR1 receptor-activating peptide (TRAP). We found that in the absence of other activators, protamine (80 µg/mL) increased the proportion of platelets with active fibrinogen receptor (binding of PAC-1) from 3.6% to 97.0% ( p < .001) measured with flow cytometry. Impedance aggregometry also increased slightly after exposure to protamine alone. When activated with ADP or TRAP protamine at 80 µg/mL reduced aggregation, from 73.8 ± 29.4 U to 46.9 ± 21.1 U ( p < .001) with ADP and from 126.4 ± 16.1 U to 94.9 ± 23.7 U ( p < .01) with TRAP. P -selectin exposure (a marker of alpha-granule release) measured by median fluorescence intensity (MFI) increased dose dependently with protamine alone, from 0.76 ± 0.20 (0 µg/mL) to 10.2 ± 3.1 (80 µg/mL), p < .001. Protamine 80 µg/mL by itself resulted in higher MFI (10.16 ± 3.09) than activation with ADP (2.2 ± 0.7, p < .001) or TRAP (5.7 ± 2.6, p < .01) without protamine. When protamine was combined with ADP or TRAP, there was a concentration-dependent increase in the alpha-granule release. In conclusion, protamine interacts with platelets in vitro having both a direct activating effect and impairment of secondary activation of aggregation by other agonists.

Background:  Administration of agents that enhance platelet reactivity may reduce the perioperative bleeding risk in patients treated with the adenosine diphosphate (ADP)-receptor antagonist ticagrelor. Adrenaline potentiates ADP-induced aggregation and activation in blood samples from ticagrelor-treated patients, but it has not previously been evaluated in vivo., Methods:  Ten healthy male subjects were included in an interventional study. A loading dose of ticagrelor (180 mg) was administered, followed 2 hours later by a gradually increased intravenous adrenaline infusion (0.01, 0.05, 0.10 and 0.15 µg/kg/min; 15 minutes at each step). Blood pressure, heart rate, platelet aggregation (impedance aggregometry), platelet activation (flow cytometry), clot formation (rotational thromboelastometry) and adrenaline plasma concentration were determined before and after ticagrelor administration and at the end of each adrenaline step., Results:  Infusion of adrenaline increased ADP-induced aggregation at all doses above 0.01 µg/kg/min. The aggregation increased from median 17 (25-75th percentiles: 14-31) to 25 (21-34) aggregation units ( p  = 0.012) at 0.10 µg/kg/min. Adrenaline infusion also increased ADP-induced fibrinogen receptor activation (from 29 [22-35] to 46 [38-57%]) and P-selectin expression (from 3.7 [3.0-4.3] to 7.7 [4.7-8.6%]), both p  = 0.012. Adrenaline infusion reduced clot formation time (97 [89-110] to 83 [76-90] seconds, p  = 0.008) and increased maximum clot firmness (59 [57-60] to 62 [61-64] mm, p  = 0.007)., Conclusion:  Infusion of adrenaline at clinically relevant doses improves in vivo platelet reactivity and clot formation in ticagrelor-treated subjects. Adrenaline could thus potentially be used to prevent perioperative bleeding complications in ticagrelor-treated patients. Studies in patients are necessary to determine the clinical importance of our observations., Trial Registry Number:  ClinicalTrials.gov NCT03441412., Competing Interests: None declared., (Georg Thieme Verlag KG Stuttgart · New York.)

Introduction: Residual pump blood from the cardiopulmonary bypass (CPB) circuit is often collected into an infusion bag (IB) and re-transfused. An alternative is to chase the residual blood into the circulation through the arterial cannula with Ringer's acetate. Our aim was to assess possible differences in hemostatic blood quality between these two techniques., Methods: Forty adult patients undergoing elective coronary artery bypass graft surgery with CPB were randomized to receive the residual pump blood by either an IB or through the Ringer's chase (RC) technique. Platelet activation and function (impedance aggregometry), coagulation and hemolysis variables were assessed in the re-transfused blood and in the patients before, during and after surgery. Results are presented as median (25-75 quartiles)., Results: Total hemoglobin and platelet levels in the re-transfused blood were comparable with the two methods, as were soluble platelet activation markers P-selectin and soluble glycoprotein VI (GPVI). Platelet aggregation (U) in the IB blood was significantly lower compared to the RC blood, with the agonists adenosine diphosphate (ADP) 24 (10-32) vs 46 (33-65), p<0.01, thrombin receptor activating peptide (TRAP) 50 (29-73) vs 69 (51-92), p=0.04 and collagen 24 (17-28) vs 34 (26-59), p<0.01. The IB blood had higher amounts of free hemoglobin (mg/L) (1086 (891-1717) vs 591(517-646), p<0.01) and D-dimer 0.60 (0.33-0.98) vs 0.3 (0.3-0.48), p<0.01. Other coagulation variables showed no difference between the groups., Conclusions: The handling of blood after CPB increases hemolysis, impairs platelet function and activates coagulation and fibrinolysis. The RC technique preserved the blood better than the commonly used IB technique.

Flow cytometry enables studies of several different aspects of platelet function in response to a variety of platelet agonists. This can be done using only a small volume of whole blood, and also in blood with low platelet counts. These properties, together with the increasing number of flow cytometers available in hospitals worldwide, make flow cytometry an interesting option for laboratories interested in studies of platelet function in different clinical settings. This review focuses on practical issues regarding the use of flow cytometry for platelet function testing. It provides an overview of available activation markers, platelet agonists, and experimental setup issues. The review summarizes previous experience and factors important to consider to perform high-quality platelet function testing by flow cytometry. It also discusses its current use and possibilities and challenges for future use of flow cytometry in clinical settings., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Human platelets express protease-activated receptor 1 (PAR1) and PAR4 but limited data indicate for differences in signal transduction. We studied the involvement of PAR1 and PAR4 in the cross-talk between thrombin and epinephrine. The results show that epinephrine acted via alpha(2A)-adrenergic receptors to provoke aggregation, secretion, and Ca(2+) mobilization in aspirin-treated platelets pre-stimulated with subthreshold concentrations of thrombin. Incubating platelets with antibodies against PAR4 or the PAR4-specific inhibitor pepducin P4pal-i1 abolished the aggregation. Furthermore, platelets pre-exposed to the PAR4-activating peptide AYPGKF, but not to the PAR1-activating peptide SFLLRN, were aggregated by epinephrine, whereas both AYPGKF and SFLLRN synergized with epinephrine in the absence of aspirin. The roles of released ATP and ADP were elucidated by using antagonists of the purinergic receptors P2X(1), P2Y(1), and P2Y(12) (i.e. NF449, MRS2159, MRS2179, and cangrelor). Intriguingly, ATP, but not ADP, was required for the epinephrine/thrombin-induced aggregation. In Western blot analysis, a low concentration of AYPGKF, but not SFLLRN, stimulated phosphorylation of Akt on serine 473. Moreover, the phosphatidyl inositide 3-kinase inhibitor LY294002 antagonized the effect of epinephrine combined with thrombin or AYPGKF. Thus, in aspirin-treated platelets, PAR4, but not PAR1, interacts synergistically with alpha(2A)-adrenergic receptors, and the PI3-kinase/Akt pathway is involved in this cross-talk. Furthermore, in PAR4-pretreated platelets, epinephrine caused dense granule secretion, and subsequent signaling from the ATP-gated P2X(1)-receptor and the alpha(2A)-adrenergic receptor induced aggregation. These results suggest a new mechanism that has ATP as a key element and circumvents the action of aspirin on epinephrine-facilitated PAR4-mediated platelet activation.

The objective of this study was to test the relative importance of the two adenosine diphosphate (ADP) receptors P2Y(1) and P2Y(12) in thrombin-induced platelet activation using specific receptor antagonists. Blood from healthy volunteers was incubated with MRS2179, a reversible P2Y(1) antagonist, or AR-C69931, a reversible P2Y(12) antagonist prior to activation with different concentrations of ADP or thrombin. Platelet function in whole blood was assayed by flow cytometry using the antibody PAC-1 to estimate the expression of conformational active alpha(IIb)beta(3), the fibrinogen receptor. Complete inhibition of P2Y(12) or P2Y(1) abolished the ADP response, but only inhibition of P2Y(12) reduced the thrombin-induced response. The relative inhibition of the thrombin response by complete inhibition of P2Y(12) was most pronounced at thrombin concentrations just enough for complete PAR1 cleavage, which is sufficient to release all ADP, giving 70-86% inhibition. Above this concentration, the relative importance of P2Y(12) inhibition decreased due to activation of ADP independent pathways. This study supports P2Y(12) as a drug target compared with P2Y(1).