Global Thrombosis Test: Occlusion is attributable to shear-induced platelet thrombus formation.

Herein, we set out a rebuttal to the publication by Claveria and co-workers published in TH Open this month entitled “Global Thrombosis Test: Occlusion by Coagulation or SIPA?” We strongly believe that the conclusions of their paper, suggesting that occlusion (OT) in the Global Thrombosis Test (GTT) is due to coagulation, rather than shear-induced platelet thrombus formation, is incorrect and the evidence and arguments they present are fundamentally flawed, with major errors both in the experimental approach and in the interpretations of the results. The evidence which they demonstrate, shows that occlusion in the GTT is, in fact, caused by high shear induced platelet thrombus formations. We set out herein the evidence for that, based on histology of the thrombus from the GTT in earlier work using electron microscopy showing large platelet aggregates, the very brief timescale of OT in the GTT compared to coagulation time and the sensitivity of the OT in the GTT to the effects of heparin, t-PA and P2Y12 inhibitors. In addition, we revisit the known pathomechanism of high shear-mediated platelet aggregation to underpin our rationale and show that the modifications to the instrument proposed by Claveria and co-authors would render the technique unphysiological. We highlight several methodological concerns and apparent misinterpreted of the data obtained. We present evidence predominantly from the authors’ own data, together with our earlier published data and evidence from the literature, showing that occlusion in the GTT occurs do to shear-induced platelet aggregation.

Remediation of ABCG5-Linked Macrothrombocytopenia With Ezetimibe Therapy

To investigate refractory hypercholesterolemia, a female patient and relatives were subjected to whole-genome sequencing. The proband was found to have compound heterozygous substitutions p. Arg446Gln and c.1118+3G>T in ABCG5, one of two genes causing sitosterolemia. When tracing these variants in the full pedigree, all maternally related heterozygotes for the intronic ABCG5 variant exhibited large platelets (over 30 fl), which segregated in an autosomal dominant manner, consistent with macrothrombocytopenia, or large platelet syndrome which may be associated with a bleeding tendency. In vitro cell-line and in vivo rat model experiments supported a pathogenic role for the variant and the macrothrombocytopenia was recapitulated in heterozygous rats and human cell lines exhibiting that single variant. Ezetimibe treatment successfully ameliorated all the symptoms of the proband with sitosterolemia and resolved the macrothrombocytopenia of the treated heterozygote relatives. Subsequently, in follow up these observations, platelet size, and size distribution were measured in 1,180 individuals; 30 were found to be clinically abnormal, three of which carried a single known pathogenic ABCG5 variant (p.Arg446Ter) and two individuals carried novel ABCG5 variants of uncertain significance. In this study, we discovered that identification of large platelets and therefore a possible macrothrombocytopenia diagnosis could easily be inadvertently missed in clinical practice due to variable instrument settings. These findings suggest that ABCG5 heterozygosity may cause macrothrombocytopenia, that Ezetimibe treatment may resolve macrothrombocytopenia in such individuals, and that increased attention to platelet size on complete blood counts can aid in the identification of candidates for ABCG5 genetic testing who might benefit from Ezetimibe treatment.

Large Platelet and Endothelial Extracellular Vesicles in Cord Blood of Preterm Newborns: Correlation with the Presence of Hemolysis

Different biomarkers are investigated to detect the causes of severe complications in preterm infants. Extracellular vesicles (EVs) are recognized as an important part of cell-to-cell communication, and their increased levels were reported in numerous pathological states. We aimed to increase our knowledge about the incidence of platelet and endothelial EVs in cord blood of preterm newborns using conventional flow cytometry. The presence of platelet (CD36+CD41+), activated platelet (CD41+CD62+), and endothelial (CD31+CD105+) EVs was analyzed. Immune electron microscopy was used to confirm the presence of EVs and the specificity of their labeling. The size of detected extracellular vesicles was in the range 400–2000 nm. The differences in the counts of EVs between the preterm and control group were not significant and no correlation of EVs count with gestation age was recorded. Cord blood plasma samples with free hemoglobin level > 1 mg/mL had more than threefold higher counts of CD36+CD41+ and CD41+CD62+ EVs (p < 0.001), while the count of CD31+CD105+ EVs was only moderately increased (p < 0.05). Further studies utilizing cytometers with improved sensitivity are needed to confirm that the analysis of large platelet and endothelial EVs mirrors the quantitative situation of their whole plasma assemblage.

SAT0241 PLATELET INDICES COULD BE SIMPLE RELIABLE PREDICTORS OF THROMBOTIC EVENTS IN PATIENTS WITH ANTIPHOSPHOLIPID SYNDROME

Background:Platelet activation is considered as a pivot pathogenic process to be responsible for thromboembolism in antiphospholipid syndrome (APS). severalstudies shown that platelet indices including platelet distribution width (PDW), mean platelet volume (MPV), large platelet rate (P-LCR) are associated with platelet activation.Objectives:This study aims to determine the correlation between platelet indices and thrombotic events in patients with APS.Methods:Platelet activation is considered as a pivot pathogenic process to be responsible for thromboembolism in antiphospholipid syndrome (APS). severalstudies shown that platelet indices including platelet distribution width (PDW), mean platelet volume (MPV), large platelet rate (P-LCR) are associated with platelet activation.Results:A total of 207 patients [135(65.2%) female, 72(34.8%) male], medianage 35(IQR 10)] was classified into thrombotic group (n=150,72.5%) and non-thrombotic group(n=57,27.5%). PDW, MPV, P-LCR were significantly higher in thrombotic group than non-thrombotic group (13.0±3.4 vs. 11.2±7.3)p<0.001, 10.7±1.4vs.10.0±3.0,p<0.001, 30.25±11.6vs. 25.1±10.4 p<0.001, respectively]. No differences in age, WBC count, hsCRP and C3 were observed between groups. Also, HGB was found to be notably higher in thrombotic group than non-thrombotic group (143±29 vs. 132±15, p=0.001).Upon univariate logistic analysis, PDW (OR 1.554, 95%CI 1.289-1.873, p<0.001), MPV (OR 1.772, 95%CI 1.268-2.476, p=0.001), P-LCR (OR 1.089, 95%CI 1.040-1.140, p<0.001) were all significantly associated with the occurrenceof thrombosis. In multivariate logistic analysis, only PDW and positive LAwere identified to be risk factors of thrombotic events (Table 1). The ROC curve showed that PDW combinedwith positive LAwas a reliable indicator of thrombotic events with an AUC of 0.796 (95%CI 0.728-0.864). The optimal cut-off value for PDW was 12.4fl with a sensitivity of 72.0% and specificity of 77.2%.Conclusion:This study confirmed that PDW, P-LCR and MPV (especially PDW) were significantly associated with thrombotic events in APS patients, which could support the theory of platelet activation being a crucial factor of thrombosis inAPS. Caution should be raised when patients with positive LA has relatively high PDW level.Disclosure of Interests:None declared

Large platelet size is associated with poor outcome in patients with metastatic pancreatic cancer

Background Platelets are a major cellular component of blood and their interaction with cancer cells is well-established to influence cancer progression and metastases. The physical size of platelets may have a critical impact on the interaction with cancer cells. In this study, we explored the potential prognostic role of platelet size measured by the determination of the mean platetlet volume (MPV) in patients with pancreatic ductal adenocarcinoma (PDAC). Methods Data from 527 patients with PDAC diagnosed and treated between 2004 and 2015 at a single center were evaluated retrospectively. Associations between MPV and baseline covariates were assessed with Wilcoxon’s rank-sum tests, χ2-tests, and Fisher’s exact tests. Median follow-up was estimated with a reverse Kaplan-Meier estimator according to Schemper and Smith. Analysis of time-to-death was performed with Kaplan-Meier estimators, log-rank tests and uni- and multivariable Cox proportional hazards models. Results The median MPV was 10.5 femto liter (fL) [9.8–11.3], ranged from 5.9 to 17.7 fL. A large platelet volume was associated with high-grade G3/4 tumors (p=0.004) and worse overall survival (OS) in patients with metastatic disease in univariable analysis (hazard ratio [HR] per fL increase in MPV=1.13 [95% CI: 1.04–1.23, p=0.005]). In multivariable analysis of metatatic PDAC patients, the adverse association between large platelets and a higher risk-of-death prevailed (adjusted HR per doubling of MPV=2.00; 95% CI: 1.10–3.62, p=0.02). Conclusions Large platelets represent a negative prognostic factor and add an independent prognostic information to well-established factors in PDAC patients. MPV should be considered for future individual risk assessment in patients with stage IV PDAC.