TL;DR
The immuno-assay for soluble P-selectin is more sensitive than the flowcytometric analysis of the percentage of P-selectin-positive cells and allowss earlier detection of platelet activation.
Platelet activation plays a central role in arterial thrombosis, thromboinflammation, and microvascular injury. Conventional platelet function tests evaluate platelet responsiveness to exogenous agonists ex vivo but do not directly reflect in vivo platelet activation. Although several soluble platelet-derived molecules, including platelet factor 4 (PF4), β-thromboglobulin (β-TG), soluble P-selectin, soluble CD40 ligand, glycocalicin, and soluble glycoprotein (GP) VI, have been investigated as in vivo platelet activation markers, their clinical use is limited by preanalytical instability, lack of platelet specificity, constitutive shedding, or uncertain disease specificity. Soluble C-type lectin-like receptor 2 (sCLEC-2) has recently emerged as a promising biomarker of in vivo platelet activation. CLEC-2 is expressed predominantly in platelets and megakaryocytes, and its soluble form is released from activated platelets as both a shed molecule and a microparticle-associated form. Compared with PF4 and β-TG, sCLEC-2 is less susceptible to artifactual release during routine blood collection, making it more suitable for clinical laboratory testing. Elevated sCLEC-2 levels have been reported in acute coronary syndrome, acute ischemic stroke, disseminated intravascular coagulation, thrombotic microangiopathy, antiphospholipid antibody syndrome, and coronavirus disease 2019 (COVID-19). In thrombocytopenic disorders, indices incorporating platelet count, such as the C2PAC index, sCLEC-2/D-dimer ratio, and sCLEC-2 × D-dimer/platelet count, may better reflect platelet activation and disease status than sCLEC-2 concentration alone. However, preanalytical standardization, assay harmonization, reference interval validation, and disease-specific cutoff values remain essential. This review summarizes the biological basis, assay systems, clinical evidence, and future perspectives of sCLEC-2 as an emerging laboratory marker of in vivo platelet activation.
Abstract Background Platelet agonists responses in vitro (i.e., reactivity) include the creation of phosphatidylserine (PS)-exposing platelets together with the activation of the fibrinogen receptors (α IIb β 3 ) and lysosomal exocytosis. It is feasible to judge the activation pathways by analyzing platelet surface annexin V, the activated fibrinogen receptor (PAC-1), and the release of lysosomal-associated membrane protein (LAMP-1), correspondingly. We postulate that, in type 2 diabetes (T2DM), surface PS of circulating platelets, unprovoked in vitro , links with whole blood (WB) agonist-induced responses. Patients and Methods After informed consent, T2DM subjects ( n = 35) were enrolled. A Percoll gradient (1.09–1.04 kg/L) separated their normal-sized platelets according to density into subpopulations ( n = 8). A flow cytometer analyzed surface annexin V (mean fluorescence intensity [MFI]) of the subfractions, unprovoked ex vivo. The datasets were subsequently correlated with platelet WB agonist-induced responses (i.e., annexin V, PAC-1, and LAMP-1 [all MFI]) to α-thrombin (10 μM), cross-linked collagen-related peptide (CRP-XL, 0.15 μg/mL), and adenosine diphosphate (ADP, 5 μM) in vitro. Results Surface annexin V (MFI) of most platelet subfractions and the magnitudes of WB agonist-induced annexin V (MFI) of normal-sized platelets in vitro associated closely. Such PS-expressing platelets also linked inversely with WB agonist-evoked surface PAC-1 (MFI) (all used agonists) and LAMP-1 (MFI) (CRP-XL, ADP only). It is concluded that surface PS, unprovoked in vitro, of most density-separated platelets connected with platelet reactivity, i.e., their WB agonist-evoked reactions in the test tube.
Platelets are anucleate cells that can be studied by benchtop flow cytometers and today there are several types of flow cytometric assays for platelets. Platelet structure studies in clinical flow cytometry mostly target rare congenital platelet glycoprotein deficiencies (e.g., Glanzmann-thrombasthenia, and Bernard-Soulier syndrome) that can be considered as the prototypes for platelet flow analysis, since the identification of complete or partial deficiency of these molecules provides the proper diagnosis. Another area of platelet flow cytometry is the detection of activated platelets. Mostly it means the gold standard platelet P-selectin assay or the investigation of PAC-1 binding-an assay where the activation dependent epitope of the GPIIb/IIIa receptor is investigated-but the measurement of platelet-leukocyte aggregates and microparticle analysis have gained increasing role. A unique platelet subtype is the subgroup designated as coated platelets that are formed by the simultaneous activation with collagen and thrombin. In clinical practice further functional assays are also informative tests: the monitoring of clopidogrel resistance by measuring the intracellular phosphorylated VASP and identifying heparin induced thrombocytopenia (HIT) by the use of patient plasma. The flow cytometric HIT test utilize the detection of phosphatidylserine expression on normal platelets or the formation of microparticles in the presence of the patient's plasma and therapeutic concentration of heparin, thus it seems reasonable to suggest it for the study of the functionality of the HIT antibodies. Nevertheless, there are also limitations to these assays as several of them require fresh samples, thus sample transport to specialized laboratories is not always feasible.
Low-titer group O whole blood (LTOWB) is increasingly used for hemorrhagic resuscitation in trauma and critical care, but the phenotype of platelets maintained within refrigerated LTOWB remains incompletely characterized. Because platelet activation may influence not only hemostatic competence but also transfusion-related inflammatory and pulmonary responses in critically ill patients, we assessed platelet surface markers in LTOWB in comparison with standard platelet concentrates and examined their evolution during 8 days of cold storage. LTOWB units prepared from qualified group O donors were stored under refrigerated conditions and studied at day 0 and day 8. Platelet phenotype was assessed by flow cytometry using CD41, CD62P, and CD63 under basal conditions and after thrombin receptor-activating peptide (TRAP) stimulation. LTOWB was compared with apheresis platelet concentrates (APC) and buffy coat-derived platelet concentrates (BC-PC). Compared with APC and BC-PC, LTOWB showed a broader distribution of CD41-positive events and a modestly lower median CD41 signal. Basal CD62P in LTOWB was lower than in APC but higher than in BC-PC, whereas basal CD63 in LTOWB was higher than in both comparator products. Between day 0 and day 8, basal CD62P and CD63 increased in LTOWB. TRAP induced marked upregulation of CD62P and CD63 at both time points; stimulated CD63 was lower at day 8 than at day 0, whereas stimulated CD62P remained high. Cold-stored LTOWB displays a distinct platelet activation phenotype characterized by progressive basal activation during refrigerated storage together with persistent agonist-inducible responses through day 8. These findings justify further functional and translational studies integrating platelet, endothelial, and lung-injury readouts to determine whether this storage-associated phenotype has consequences for microvascular hemostasis, immunothrombosis, or transfusion-associated pulmonary complications after hemorrhagic resuscitation.
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