To Örebro University

BACKGROUND: Given the role of platelets in coronary artery disease (CAD), assessment of a soluble platelet-activation marker may be useful to improve thrombotic risk stratification.

OBJECTIVES: This study aimed to perform a meta-analysis investigating the association between levels of 14 such markers associated with CAD.

METHODS: PubMed, Web of Science, and Excerpta Medica dataBASE (EMBASE) were searched until November 2024. The primary end point was the difference in levels of 11-dehydro-thromboxane B2, 2,3-dinor-thromboxane B2, β-thromboglobulin, soluble CD40L (sCD40L), glycocalicin, glycoprotein (GP)V, GPVI, matrix metalloproteinase (MMP)-9 and MMP-2, platelet factor (PF)4, soluble (s) P-selectin, SCUBE1, serotonin and thrombospondin (TSP)-1 between patients with CAD and healthy subjects (HSs) in plasma and/or serum. When possible, patients with CAD were stratified into acute coronary syndrome (ACS) and chronic coronary disease. Standardized mean difference (SMD) was calculated.

RESULTS: Due to heterogeneity in the assessed studies, meta-analysis was performed for sCD40L, soluble GPV, MMP-9, PF4, sP-selectin, SCUBE1, and TSP-1. All markers but TSP-1 were significantly elevated in patients with CAD compared with HSs. Differences in sCD40L and SCUBE1 were statistically significant only when studies that assessed plasma were combined with those that assessed serum. When compared with HSs, the differences were bigger in patients with ACS than patients with chronic coronary disease for MMP-9 (SMD, 2.49 vs 0.49), PF4 (SMD, 2.01 vs 0.96), and sP-selectin (SMD, 1.81 vs 0.63). Publication bias was identified for sCD40L and, in ACS, for sP-selectin and PF4.

CONCLUSION: The increased levels of sCD40L, soluble GPV, MMP-9, PF4, sP-selectin, and SCUBE1 in patients with CAD compared with HSs provide a rationale for designing new studies to address the potential of such molecules as biomarkers for thrombotic risk stratification.

Background: Platelets contain many heterogeneous carbohydrates (glycans), often capped by sialic acid. The removal of sialic acid (desialylation) is important for platelet function and clearance, leading to novel diagnostic markers. Platelet desialylation can be easily measured using inexpensive, user-friendly lectins, and flow cytometry.

Objectives: Here, the Platelet Physiology Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis (ISTH) carried out a survey to assess current methods used for platelet desialylation. Based on the survey results, a consensus protocol was drafted and tested.

Methods: A survey/questionnaire was posted on the ISTH Platelet Physiology Standardization Committee pages. Washed platelets and diluted apheresis platelets were diluted to 50 and 200 x 106/mL +/- CaCl2. Platelets were stained with a concentration range of either beta-galactose binding fluoresceine-conjugated lectin Ricinus communis agglutinin 1 (RCA-1) or Erythrina cristagalli lectin (ECL). As positive controls, different recombinant sialidases were tested.

Results: The results of the survey (N = 20) showed that flow cytometry and RCA-1 are mostly used to assess platelet desialylation. Calcium did not significantly influence lectin binding, and optimal binding was achieved with ECL and RCA-1 at 2 and 5 mu g/mL, respectively. The specificity of lectins varied, particularly after sialidase treatment, compared with cold-stored platelets. These findings contribute to the standardization of desialylation measurements, particularly in patient samples.

Conclusion: Our findings demonstrate that flow cytometry using RCA-1 and ECL is a robust method for quantifying platelet desialylation. The proposed standardized protocol addresses key preanalytical variables, enabling reproducible and accurate analysis of platelet glycosylation.

Over the past 3 decades, omics technologies have revolutionized our understanding of platelet molecular content and organization, enabling the systematic analyses of platelet physiology. Among these approaches, proteomics has been especially significant in discovering as well as validating molecular mechanisms of platelet function in health and disease. However, several conceptual and practical challenges continue to limit the full utility of platelet proteomics tools and data. Methodological and analytical inconsistencies remain a key concern, with biological and technical variables exerting substantial influence on study outcomes and interpretation. These issues are compounded by the rapid pace of proteomics tool development and dataset collection, outstripping efforts to standardize best practices and ensure consensus, as platelet proteomics consolidates itself as a tool for research even outside the thrombosis and hemostasis field. In this communication from the International Society on Thrombosis and Haemostasis Scientific and Standardization Committee, we highlight recent advances in platelet proteomics studies, and we identify where collective efforts can strengthen experimental design, execution, and analysis. As a practical recommendation, we encourage platelet biologists to recognize current discrepancies and advance efforts to standardize and customize methods and reporting practices, including blood collection, platelet isolation, data acquisition, and data interpretation. By aligning protocols and ensuring detailed reporting, the field can more effectively integrate proteomics findings and accelerate our understanding of platelet biology.

Introduction: During cardiac surgery, dosing of protamine to reverse heparin after cardiopulmonary bypass is delicate. Overdosing of protamine with negative effects on coagulation1 and platelet function2,3 must be balanced against underdosing with inadequate neutralization of heparin. Modern protamine titration equipments have led to lower doses ofprotamine being used.4 We studied possible remaining or rebound heparin activity after reversal with protamine using a commercial titration system.

Methods: Forty elective CABG patients were included with blood samples collected at arrival to the intensive care unit (ICU) and three hours after arrival to the ICU. The dose of heparin and protamine was calculated by a titration system using a dose response curve. The need for additional protamine was assessed with the titration system at the end of surgery, and additional protamine was given if heparin was detected. Presence of heparin at ICU arrival and at 3 hours was measured by anti-factor Xa (anti-FXa), thrombin time (TT) and aPTT. Coagulation capacity was assessed with endogenous thrombin potential (ETP). Postoperative bleeding was registered after 3 hours and after drain removal. Data are presented as mean ± SD or median and interquartile range (IQR) depending on normality. Association of variables was assessed with Spearman’s rank correlation and linear regression. For categorical variables Fishers exact test was used and for comparison of groups the Wilcoxon test wasused.

Results: The patients received 30 800 ± 6 300 units (U) of heparin. The mean protamine/heparin ratio (P/H ratio) was 0.54 (±0.14) mg/100 U, with a range from 0.25 to 0.84 mg/100 U. At arrival to the ICU (81 (IQR 34) minutes after protamine) 63% of the patients had measurable anti-FXa levels, and median anti-FXa was 0.09 (IQR 0.25) U/mL. Median TT at ICU arrival was 19.7 (IQR 6.4) seconds (s), aPTT was 27.5 (IQR 8.0) s and ETP 603 (IQR 899) U. Anti-FXa correlated significantly to both APTT (r=0.50, p=0.0018) and TT (r=0.66, p<0.001). ETP correlated negatively to anti-FXa (r=-0.57, p<0.001). There was an inverse relation between the P/H ratio and TT (r=-0.48; p=0.004) and aPTT (r=-0.50; p=0.0015) at ICU arrival. There was also an inverse relation between P/H ratio and anti-FXa (r=-0.32; p=0,054) and a positive correlation between P/H ratio and ETP (r=0.41; p=0.012) (Figure 1). Three hours after ICU arrival all but one of the patients had detectable levels of anti-FXa (p<0.001), and over-all anti-FXa had increased slightly to 0.17 (IQR 0.15) U/mL (p=0.11 vs ICU arrival). TT had increased to 22.9 (IQR 8.0) s (p=0.019) and ETP dropped to 215 (IQR 403) U (p<0.001). At this time point however there was no correlation between P/H ratio, heparin or protamine dose on one hand and heparin activity (anti-FXa, TT) or coagulation potential ETP on the other. The total postoperative bleeding was 623 (IQR 290) mL of which 185 (IQR 140) mL was during the first three hours. Bleeding during the first 3 hours did not correlate to P/H ratio or heparin markers and thrombin generation at ICU arrival.

Conclusions: At ICU arrival, 81 minutes after protaminization, most patients showed remaining heparin activity. This activity was directly related to prior dose of protamine. Heparin activity increased during the following 3 hours but did not correlate to protamine dosing or bleeding. The study is consistent with prior data describing reappearance of heparin in the postoperative period independent of the dosing of protamine5. The lack of relation between postoperative heparin activity and postoperative bleeding underlines that other factors than remaining or rebound heparin plays an important role for postoperative bleeding after cardiac surgery.

References:

Protamine sulfate down-regulates thrombin generation by inhibiting factor V activation. 114(8):1658-1665. 2009

Protamine stimulates platelet aggregation in vitro with activation of the fibrinogen receptor and alpha-granule release, but impairs secondary activation via ADP and thrombin receptors. 32(1):90-96. 2021

Platelet Function is Preserved After Moderate Cardiopulmonary Bypass Times But Transiently Impaired After Protamine. 37(7):1110-1120. 2023

Anticoagulation management during multivessel coronary artery bypass grafting: a randomized trial comparing individualized heparin management and conventional hemostasis management. 13(7):1196-1206. 2015

Reappearance of circulating heparin in whole blood heparin concentration-based management does not correlate with postoperative bleeding after cardiac surgery. 28(4): 1003-1007. 2014

Genome-wide platelet transcriptomics is increasingly used to uncover new aspects of platelet biology and as a diagnostic and prognostic tool. Nevertheless, platelet isolation methods for transcriptomic studies are not standardized, introducing challenges for cross-study comparisons, data integration, and replication. In this prospective multicenter study, called "Standardizing Platelet Transcriptomics for Discovery, Diagnostics, and Therapeutics in the Thrombosis and Hemostasis Community (STRIDE)" by the ISTH SSCs, we assessed how three of the most commonly used platelet isolation protocols influence metrics from next-generation bulk RNA sequencing and functional assays. Compared with washing alone, more stringent removal of leukocytes by anti-CD45 beads or PALLTM filters resulted in a sufficient quantity of RNA for next-generation sequencing and similar quality of RNA sequencing metrics. Importantly, stringent removal of leukocytes resulted in the lower relative expression of known leukocyte-specific genes and the higher relative expression of known platelet-specific genes. The results were consistent across enrolling sites, suggesting the techniques are transferrable and reproducible. Moreover, all three isolation techniques did not influence basal platelet reactivity, but agonist-induced integrin αIIbβ3 activation is reduced by anti-CD45 bead isolation compared to washing alone. In conclusion, the isolation technique chosen influences genome-wide transcriptional and functional assays in platelets. These results should help the research community make informed choices about platelet isolation techniques in their own platelet studies.

Background: Platelet count alone does not reliably predict bleeding risk, suggesting platelet function is important to monitor in patients with thrombocytopenia. There is still an unmet need for improved platelet function diagnostics in patients with low platelet count in many clinical situations. Flow cytometry is a promising tool allowing reliable platelet function study in this setting.

Objectives: The goal of this joint project between the International Society on Thrombosis and Haemostasis (ISTH) Scientific Standardization Committee (SSC) Subcommittees on Platelet Physiology and Platelet Immunology is to provide expert consensus guidance on the use of flow cytometry for the evaluation of platelet function, particularly activation, in patients with low platelet counts.

Methods: A literature review was performed to identify relevant questions and areas of interest. An electronic expression of interest form was thereafter announced on the ISTH webpage, followed by a survey encompassing 37 issues regarding preanalytical, analytical, postanalytical, and performance aspects. Areas of disagreement or uncertainty were identified and formed the basis for 2 focus group discussions.

Results: Consensus recommendations relative to patient sample collection, preanalytical variables, sample type, platelet-count cutoff, any potential specific modification of the standard flow cytometry protocol, and results expression and reporting are proposed based on the current practices of experts in the field as well as on literature review.

Conclusion: The proposed consensus recommendations would allow standardization of protocols in upcoming clinical studies. The clinical utility of platelet function testing using flow cytometry to predict bleeding risk still needs rigorous multicenter outcome studies in patients with thrombocytopenia.

BACKGROUND: Procoagulant platelets are a subpopulation of highly activated platelets that promote coagulation through surface-exposed, negatively charged phospholipids, especially phosphatidylserine (PS). Procoagulant platelets are important for clot stabilization during haemostasis and an increased number of these platelets is associated with thrombotic risk. There is a need for harmonisation in this area since many of the markers and methods used to assess procoagulant platelets are not specific when used in isolation but are also associated with platelet apoptosis.

OBJECTIVE: We initiated this project to identify a minimum set of markers and/or methods that can detect and distinguish procoagulant platelets from apoptotic platelets.

METHODS AND RESULTS: The study design involved a primary panel with twenty-seven international experts participating in an online survey and moderated virtual focus group meetings. Primary and secondary panel members were then invited to provide input on themes and statements generated from the focus groups. This led to a recommendation to use flow cytometry and a combination of the following three surface markers to differentiate procoagulant from apoptotic platelets: P-selectin (CD62P), PS (recognized by annexin V), and a platelet-specific receptor GPIX (CD42a) or αIIb integrin (CD41, GPIIb).

CONCLUSION: Procoagulant platelets are expected to be positive for all three markers, while apoptotic platelets will be positive for annexin V and the platelet specific surface receptor(s) but negative for P-selectin.

Platelets are transfused to patients to prevent bleeding. Since both preparation and storage can impact the hemostatic functions of platelets, we studied platelet concentrates (PCs) with different initial composition in regard to platelet fragmentation and its impact on storage-induced changes in activation potential. Ten whole blood derived PCs were assessed over 7 storage days. Using flow cytometry, platelet (CD41+) subpopulations were characterized for activation potential using activation markers (PAC-1, P-selectin, and LAMP-1), phosphatidylserine (Annexin V), and mitochondrial integrity (DiIC1(5)). Aggregation response, coagulation, and soluble activation markers (cytokines and sGPVI) were also measured. Of the CD41+ events, the PCs contained a median of 82% normal-sized platelets, 10% small platelets, and 8% fragments. The small platelets exhibited procoagulant hallmarks (increased P-selectin and Annexin V and reduced DiIC1(5)). Normal-sized platelets responded to activation, whereas activation potential was decreased for small and abolished for fragments. Five PCs contained a high proportion of small platelets and fragments (median of 28% of CD41+ events), which was significantly higher than the other five PCs (median of 9%). A high proportion of small platelets and fragments was associated with procoagulant hallmarks and decreased activation potential, but, although diminished, they still retained some activation potential throughout 7 days storage.

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 × 109/g to 1.91 ± 0.51 × 109/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.

Arterial thrombosis (AT) originates through platelet-mediated thrombus formation in the blood vessel and can lead to heart attack, stroke, and peripheral vascular diseases. Restricting the thrombus growth and its simultaneous monitoring by visualisation is an unmet clinical need for a better AT prognosis. As a proof-of-concept, we have engineered a nanoparticle-based theranostic (combined therapy and monitoring) platform that has the potential to monitor and restrain the growth of a thrombus concurrently. The theranostic nanotool is fabricated using biocompatible super-paramagnetic iron oxide nanoparticles (SPIONs) as a core module tethered with the anti-platelet agent Abciximab (ReoPro) on its surface. Our in vitro feasibility results indicate that ReoPro-conjugated SPIONS (Tx@ReoPro) can effectively prevent thrombus growth by inhibiting fibrinogen receptors (GPIIbIIIa) on the platelet surface, and simultaneously, it can also be visible through non-invasive magnetic resonance imaging (MRI) for potential reporting of the real-time thrombus status.