In flow cytometry, individual cells are investigated. Platelet activation is normally reported in form of percentage of platelets expressing the marker (positive platelets) and/or mean/median fluorescence intensity (MFI) for the entire analyzed population. None of these take into account the variance of the marker expression between individual platelets. This can be obtained as data on coefficient of variation (CV). This study explores if CV provides additional information regarding platelet function. Samples from platelet concentrates (PCs) prepared by apheresis- (n = 13) and interim platelet unit (IPU) technique (n = 26) and stored for 6-7 days were included and compared. Spontaneous- and agonist-induced expression of activation markers (fibrinogen binding and exposure of P-selectin, LAMP-1, and CD63) was investigated as percentage positive platelets, MFI and CV. Spontaneous expression of P-selectin as percentage positive platelets and MFI was higher for IPU PCs than apheresis PCs, which in contrast had higher agonist-induced activation. CV for spontaneous fibrinogen binding and P-selectin exposure was larger for apheresis PCs, while IPU PCs generally had larger CV for P-selectin, LAMP-1, and CD63 after agonist stimulation. Our findings show that CV adds additional information when assessing platelet activation by providing data on the variation in activation responses within the platelet population.

In X-linked thrombocytopenia with thalassemia (XLTT; OMIM 314050), caused by the mutation p.R216Q in exon 4 of the GATA1 gene, male hemizygous patients display macrothrombocytopenia, bleeding diathesis and a ß-thalassemia trait. Herein, we describe findings in two unrelated Swedish XLTT families with a bleeding tendency exceeding what is expected from the thrombocytopenia. Blood tests revealed low P-PAI-1 and P-factor 5, and elevated S-thrombopoietin levels. Transmission electron microscopy showed diminished numbers of platelet a- and dense granules. The proteomes of isolated blood platelets from 5 male XLTT patients, compared to 5 gender- and age matched controls, were explored. Quantitative mass spectrometry showed alterations of 83 proteins (fold change =±1.2, q< .05). Of 46 downregulated proteins, 39 were previously reported to be associated with platelet granules. Reduced protein levels of PTGS1 and SLC35D3 were validated in megakaryocytes of XLTT bone marrow biopsies by immunohistochemistry. Platelet function testing by flow cytometry revealed low dense- and a-granule release and fibrinogen binding in response to ligation of receptors for ADP, the thrombin receptor PAR4 and the collagen receptor GPVI. Significant reductions of a number of a-granule proteins overlapped with a previous platelet proteomics investigation in the inherited macrothrombocytopenia gray platelet syndrome (GPS). In contrast, Ca2+ transporter proteins that facilitate dense granule release were downregulated in XLTT but upregulated in GPS. Ingenuity Pathway Analysis showed altered Coagulation System and Protein Ubiquitination pathways in the XLTT platelets. Collectively, the results revealed protein and functional alterations affecting platelet a- and dense granules in XLTT, probably contributing to bleeding.

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 mu g/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 mu g/mL) increased the proportion of platelets with active fibrinogen receptor (binding of PAC-1) from 3.6% to 97.0% (p amp;lt; .001) measured with flow cytometry. Impedance aggregometry also increased slightly after exposure to protamine alone. When activated with ADP or TRAP protamine at 80 mu g/mL reduced aggregation, from 73.8 +/- 29.4 U to 46.9 +/- 21.1 U (p amp;lt; .001) with ADP and from 126.4 +/- 16.1 U to 94.9 +/- 23.7 U (p amp;lt; .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 mu g/mL) to 10.2 +/- 3.1 (80 mu g/mL), p amp;lt; .001. Protamine 80 mu g/mL by itself resulted in higher MFI (10.16 +/- 3.09) than activation with ADP (2.2 +/- 0.7, p amp;lt; .001) or TRAP (5.7 +/- 2.6, p amp;lt; .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: Although flow cytometry is often brought forward as a preferable method in the setting of thrombocytopenia, the relative effects of low sample counts on results from flow cytometry-based platelet function testing (FC-PFT) in comparison with light transmission aggregometry (LTA) and multiple electrode aggregometry (MEA) has not been reported. Objectives: To compare the effects of different sample platelet counts (10, 50, 100, and 200x10(9)L(-1)) on platelet activation measured with FC-PFT, LTA, and MEA using the same anticoagulant and agonist concentrations as for the commercial MEA test. Methods: Platelets were stimulated with two commonly used platelet agonists (ADP [6.5 mu molL(-1)] and PAR1-AP [TRAP, 32 mu molL(-1)]). The specified sample platelet counts were obtained by combining platelet-rich and platelet poor hirudinized plasma in different proportions with or without red blood cells. Results: For FC, P-selectin exposure and PAC-1 binding was reduced at 10x10(9)L(-1) after stimulation with PAR1-AP (by approximately 20% and 50%, respectively), but remained relatively unchanged when ADP was used as agonist (n=9). The platelet count-dependent effects observed with PAR1-AP were eliminated when samples were pre-incubated with apyrase, implying that reduced purinergic signaling was the main underlying factor (n=5). Both aggregometry-based PFTs showed a 50% reduction at 50x10(9)L(-1) and more than 80% reduction at 10x10(9)L(-1), irrespective of agonist used (n=7). Conclusions: Although FC-PFT is generally preferable to aggregometry-based PFTs in situations with low sample platelet counts, a careful optimization of experimental parameters is still required in order to eliminate platelet count-related effects.

Temporarily improved platelet reactivity may reduce the bleeding in patients on antiplatelet therapy who have ongoing bleeding or who are in need of acute surgery. Adrenaline can bind to adrenergic a2A-receptors on platelets and potentially enhance platelet reactivity.

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.

Platelet function disorders (PFDs) are common in patients with mild bleeding disorders (MBDs), yet the significance of laboratory findings suggestive of a PFD remain unclear due to the lack of evidence for a clinical correlation between the test results and the patient phenotype. Herein, we present the results from a study evaluating the potential utility of platelet function testing using whole-blood flow cytometry in a cohort of 105 patients undergoing investigation for MBD. Subjects were evaluated with a test panel comprising two different activation markers (fibrinogen binding and P-selectin exposure) and four physiologically relevant platelet agonists (ADP, PAR1-AP, PAR4-AP, and CRP-XL). Abnormal test results were identified by comparison with reference ranges constructed from 24 healthy controls or with the fifth percentile of the entire patient cohort. We found that the abnormal test results are predictive of bleeding symptom severity, and that the greatest predictive strength was achieved using a subset of the panel, comparing measurements of fibrinogen binding after activation with all four agonists with the fifth percentile of the patient cohort (p=0.00008, hazard ratio 8.7; 95% CI 2.5-40). Our results suggest that whole-blood flow cytometry-based platelet function testing could become a feasible alternative for the investigation of MBDs. We also show that platelet function testing using whole-blood flow cytometry could provide a clinically relevant quantitative assessment of platelet-related hemostasis.

It is recognised that platelets respond differently to activation, where a subpopulation of platelets adopt a procoagulant phenotype while others are aggregatory. However, it has not been thoroughly tested whether these subpopulations will remain in maximally activated samples, or if they are merely a result of different platelet sensitivities to agonist activation. Here platelets were activated with gradually increasing concentrations of thrombin and/or the GPVI agonist cross-linked collagen-related peptide (CRP-XL). Platelet activation was investigated using a novel six-colour flow cytometry protocol evaluating exposure of phosphatidylserine, active conformation of the fibrinogen receptor alpha(IIb)beta(3), alpha-granule and lysosomal release (P-selectin and LAMP-1 exposure), mitochondrial membrane integrity and platelet fragmentation. Upon activation by CRP-XL or thrombin+CRP-XL, platelets formed three differently sized subpopulations. Normal-sized platelets showed high exposure of aggregatory active alpha(IIb)beta(3) and intact mitochondria, while the smaller platelets and platelet fragments showed high exposure of procoagulant phosphatidylserine. The distribution of platelets between the differently sized subpopulations remained stable despite high agonist concentrations. All three were still present after 30 and 60 min of activation, showing that all platelets will not have the same characteristics even after maximal stimulation. This suggests that platelet subpopulations with distinct activation patterns exist within the total platelet population.

Platelet-derived polyphosphates (polyP), stored in dense granule and released upon platelet activation, have been claimed to enhance thrombin activation of coagulation factor XI (FXI) and to activate FXII directly. The latter claim is controversial and principal results leading to these conclusions are probably influenced by methodological problems. It is important to consider that low-grade contact activation is initiated by all surfaces and is greatly amplified by the presence of phospholipids simulating the procoagulant membranes of activated platelets. Thus, proper use of inhibitors of the contact pathway and a careful choice of materials for plates and tubes is important to avoid artefacts. The use of phosphatases used to degrade polyP has an important drawback as it also degrades the secondary activators ADP and ATP, which are released from activated platelets. In addition, the use of positively charged inhibitors, such as polymyxin B, to inhibit polyP in platelet-rich plasma and blood is problematic, as polymyxin B also slows coagulation in the absence of polyP. In conclusion we hope awareness of the above caveats may improve research on the physiological roles of polyP in coagulation. © 2016 Authors; published by Portland Press Limited.

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