Flow Cytometric Detection of Procoagulant Properties of Plasma from Patients with Clinically Confirmed Vaccine-Induced Immune Thrombotic Thrombocytopenia

Background: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a severe prothrombotic complication of adenoviral vaccines including ChAdOx1 nCoV-19 (AstraZeneca) vaccine. The putative mechanism involves formation of pathological anti-PF4 antibodies that activate platelets via the FcγRIIa receptor to drive thrombosis and the associated thrombocytopenia. Functional assays are important in the VITT diagnostic pathway as not all detectable PF4 antibodies are pathogenic. Detection of procoagulant platelets (platelets supporting thrombin generation) in presence of PF4 has been proposed as a diagnostic assay for VITT (Althaus et al). Procoagulant platelets are not typically generated in response to low level agonist stimulation; however, combination of ligand binding of G-protein coupled receptors (GPCR) (eg. PAR1) and ITAM linked receptors (eg. GPVI, CLEC2 and FcγRIIa) synergistically induce procoagulant platelet formation. Here, we describe an alternative flow cytometric assay to diagnose VITT. We hypothesized that priming of platelets with a PAR1 agonist at a level sufficient to release PF4, but insufficient to generate a significant procoagulant response in donor platelets, would provide a platform in which procoagulant response would be dependent on presence of FcγRIIa dependent procoagulant antibodies in patient plasma, without requirement for additional PF4. Methods: Our previously established flow cytometry-based procoagulant platelet assay (using cell death marker GSAO and P-selectin) was modified to incorporate exogenous patient plasma and performed on whole blood from healthy donors screened for FcγRIIa responsiveness (aggregation response to anti-CD9 antibody, ALB6), primed with 5 μM SFLLRN. The assay was performed on Australian patients referred for confirmatory VITT testing with probable VITT (confirmed thrombosis within 4-42 days of ChAdOx1 nCov-19 vaccination, D-Dimer > 5x ULN, platelets < 150 x 10 9/L or falling platelet count) after screening on PF4/heparin ELISA (Asserachrom HPIA IgG Assay, Stago Diagnostics). Procoagulant response was also measured in presence of 0.5 U/mL and 100 U/mL heparin, monoclonal FcγRIIa blocking antibody, IV.3, and intravenous immunoglobulin, IVIg. Some plasmas were incubated with ChAdOx1 nCoV-19 or SARS-CoV-2 spike protein. Flow cytometry positive patients were also tested by serotonin release assay (SRA) and multiplate aggregometry. Clinical correlation was obtained. Results: Citrated plasma from 49 unique patients with suspected VITT are reported. Plasma from ELISA+ve patients with clinical picture consistent with VITT (n=31), significantly increased the procoagulant platelet proportions in healthy donors in presence of 5 μM SFLLRN (p<0.0001, Figure 1A). This increase was not seen with plasma from healthy donors (n=14); or individuals exposed to ChAdOx1 nCov-19 vaccine without VITT: thrombocytopenic thrombosis patients who were ELISA-ve and SRA-ve (n=14); or low-level ELISA+ve patients without thrombocytopenia who were negative by either multiplate or SRA (n=4). The procoagulant platelet response induced by VITT positive plasma was reduced with low dose heparin (0.5 U/mL, p<0.01) except for 3 patients who showed a heparin-enhancing effect (Figure 1B). High dose heparin (100 U/mL, p<0.0001), IV.3 (10 µg/mL, p<0.0001) or IVIg (10 mg/mL, p<0.0001) abolished the procoagulant response (Figures 1C-D). The in vitro effect of IVIg was predictive of the in vivo response to IVIg therapy (Figure 1E). Addition of SARS-CoV-2 spike protein had no effect on the procoagulant platelet response. ChAdOx1 nCov-19 had an inconsistent effect on procoagulant platelet formation in presence of VITT plasma. Use of donors without a robust aggregation response to ALB6 resulted in false negative results. Conclusion: Induction of FcγRIIa dependent procoagulant response by patient plasma, suppressible by high dose heparin and IVIg, is highly indicative of VITT in the correct clinical circumstance. This assay modification of priming donor platelets from known FcγRIIa responsive donors with a GPCR agonist to potentiate the ITAM signaling from platelet activating immune complexes, results in a sensitive and specific assay. This may represent a functional platform that can be adopted into diagnostic laboratories to identify patients with platelet-activating antibodies and potentially predict treatment responses. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Generic Clopidogrel: Has Substitution for Brand Name Plavix® Been Effective?

Background: Following the expiration of brand name exclusivity of Plavix® in 2012, generic clopidogrel bisulfate was approved. As a widely prescribed medication with significant inter-patient pharmacokinetic and pharmacodynamic variability, data regarding the impact of switching to generic clopidogrel bisulfate on patients is needed. Objective: The objective of this study was to determine whether generic clopidogrel bisulfate is as efficacious as Plavix® for the inhibition of platelet aggregation. Methods: Patients treated with Plavix® monotherapy (n = 254) or generic clopidogrel bisulfate monotherapy (n = 185) were included in this retrospective review. Confounding factors previously found to affect clopidogrel responsiveness (diabetes, female sex, and smoking) were assessed, as well as medications classified as substrates, inducers, and inhibitors of enzymes involved in clopidogrel metabolism. Whole blood impedance aggregometry was used to measure platelet aggregation in response to adenosine diphosphate. Patients were tested after ≥2 weeks of treatment and designated as non-responders if aggregation response exceeded sensitivity thresholds of 6 ohms of impedance. Results: The introduction of generic clopidogrel bisulfate was associated with a decrease in antiplatelet resistance (44% to 31%, p < 0.01) and decreased mean ohms of resistance (5.06 ± 4.55 to 3.32 ± 4.03, p < 0.01). Prior to analysis of secondary outcomes, 217 patients were eliminated due to antiplatelet usage for longer than 3 years (n = 123 for Plavix® and n = 118 for clopidogrel). There was no statistically significant finding in prevalence of secondary events. Conclusion: Resistance rates to the antiplatelet, clopidogrel are significantly lower since the switch to generic formulations. Further investigation into the impact of variability between clopidogrel bisulfate formulations is needed.

Pharmacodynamic Comparison of Ticagrelor Monotherapy Versus Ticagrelor and Aspirin in Patients After Percutaneous Coronary Intervention: The TEMPLATE (Ticagrelor Monotherapy and Platelet Reactivity) Randomized Controlled Trial

Background To assess differences in platelet inhibition during ticagrelor monotherapy (TIC) or dual therapy with ticagrelor and aspirin (TIC+ASP) in patients after percutaneous coronary intervention using a comprehensive panel of functional tests. Methods and Results In a single‐center parallel group, open label, randomized controlled trial, 110 participants were randomized to receive either TIC (n=55) or TIC+ASP (n=55) for 4 weeks. The primary outcome was the platelet aggregation response with 10 μmol/L thrombin receptor activation peptide‐6 (TRAP‐6). The secondary outcomes were platelet aggregation responses and binding of surface activation markers with a panel of other activators. The mean percentage aggregation for 10 μmol/L TRAP‐6 was similar for the TIC and TIC+ASP groups (mean difference+4.29; 95% CI, −0.87 to +9.46). Aggregation was higher in the TIC group compared with the TIC+ASP group with 1 μg/mL (+6.47; +2.04 to +10.90) and 0.5 μg/mL (+14.00; +7.63 to +20.39) collagen related peptide. Aggregation responses with 5 μmol/L TRAP‐6, 5 μmol/L or 2.5 μmol/L thromboxane A 2 receptor agonist and surface activation marker binding with 5 μmol/L TRAP‐6 or 0.5 μg/mL collagen related peptide were the same between the treatment groups. Conclusions Patients with PCI show similar levels of inhibition of most platelet activation pathways with TIC compared with dual therapy with TIC + ASP. However, the greater aggregation response with collagen related peptide during TIC indicates incomplete inhibition of glycoprotein VI (collagen) receptor‐mediated platelet activation. This difference in pharmacodynamic response to anti‐platelet medication may contribute to the lower bleeding rates observed with TIC compared with dual antiplatelet therapy in recent clinical trials. Registration Information URL: https://www.isrctn.com ; Unique Identifier ISRCTN84335288.

“Tuning aggregative versus non-aggregative lectin binding with glycosylated nanoparticles by the nature of the polymer ligand”

Varying the nature of polymeric linkers modulates the aggregation response of glycosylated nanoparticles upon interaction with lectins.

High Prevalence of Sticky Platelet Syndrome in Patients with Infertility and Pregnancy Loss

Platelet hyperaggregability, known as sticky platelet syndrome (SPS), is a prothrombotic disorder that has been increasingly associated with pregnancy loss. In this retrospective study, we aimed to investigate the clinical and diagnostic relevance of SPS in 208 patients with infertility and unexplained pregnancy loss history. We studied 208 patients that had been referred to undergo a dose-dependent platelet aggregation response to adenosine diphosphate and epinephrine using light transmission aggregometry modified by Mammen during an 11-year period. Patients’ platelet aggregation response was compared with platelet function in 29 female healthy controls of fertile age with no previous history of pregnancy loss. We found a prevalence of SPS type II (33.2%) in 208 female patients with infertility and pregnancy loss. ∆-epinephrine-induced platelet aggregation in patients with SPS was significantly decreased (median 7% and range −21 to 43%) compared to patients without SPS (median 59%, range 7–88% and p < 0.0001) and healthy controls (median 57%, range 8–106% and p < 0.0001). The optimum SPS-diagnostic cutoff value for ∆-epinephrine aggregation was ≤32% (sensitivity 95.7%, specificity 95.2%). SPS patients with low-dose acetylsalicylic acid (ASA) therapy (n = 56) showed improved pregnancy outcome (32 pregnancies; live births n = 18 (56%)) compared to SPS patients without low-dose ASA (n = 13) (3 pregnancies; live births n = 1 (33%)). Our study demonstrates the clinical and diagnostic relevance of platelet hyperaggregation in women with infertility and pregnancy loss history. Further studies should investigate the potential of SPS as a novel decisional tool with both diagnostic and clinical implications in infertility and pregnancy loss.

Comparing a New Bovine Source Heparin to the Clinically Used Porcine Heparin for Platelet Function Effects and Heparin-Induced Thrombocytopenia Potential

Background Heparin is a sulfated polysaccharide obtained from intestinal mucosa with anticoagulant properties that is widely used as a standard clinical therapeutic agent to treat and prevent thrombosis. Heparin is known to affect platelet function, and among its side effects is heparin-induced thrombocytopenia (HIT) that can occur in about 1% of patients exposed to heparin. Presently, only porcine source heparin is approved for use in the United States. The aims of this study were to determine if platelet activation by physiological agonists and platelet aggregation induced by HIT antibodies would be equivalent in the presence of bovine source heparin and porcine source heparin. Materials and Methods Seven lots of bovine heparin from Eurofarma and 3 lots of commercial clinical grade porcine heparin (Pfizer/Hospira) were evaluated. The USP Reference Standard for porcine heparin was used to determine anti-Xa and anti-IIa potencies of the bovine heparins. For each study, blood was collected from healthy volunteers (n=5 per test group), anticoagulated with sodium citrate, and centrifuged to obtain platelet rich plasma (PRP). Platelet aggregation responses were assessed using the BioData PAP-8 platelet aggregometer. For the first aim to evaluate platelet function, PRP was combined with heparin at final concentrations of 10.0, 1.0, and 0.1 µg/mL, covering both therapeutic and prophylactic ranges. Platelet agonists included adenosine diphosphate (ADP), collagen, epinephrine, arachidonic acid, and thrombin receptor agonist peptide (TRAP). The aggregation response was quantitated in terms of primary slope (PS), area under the curve (AUC), maximum aggregation (MA), and final aggregation (FA). For the second aim to evaluate the HIT potential, antibodies to the complex of heparin-platelet factor 4 (H-PF4) from banked HIT patient apheresis fluid were combined with donor PRP and heparin. Heparins were tested at final concentrations of 0.1, 0.4, 0.8, 1, and 100 U/mL. PS and FA results were recorded. For all data, comparisons were analyzed with 2-Way ANOVA using SigmaPlot software. Results In the presence of either bovine (BMH) or porcine heparin (PMH), the normal platelet aggregation response of all donors was not altered from that obtained with saline (see representative aggregation tracing in the image below). All heparin concentrations produced the same response. There were no significant differences between the bovine and porcine heparins for each of the 4 platelet aggregation parameters for ADP, arachidonic acid, collagen, epinephrine, and TRAP. Variation in the PS for arachidonic acid and collagen need to be assessed in a larger pool of donors to assure the lack of significant difference. Platelet activation to H-PF4 antibodies was strong at 0.1 to 1 U/mL concentrations with the expected inhibition observed when using 100 U/mL heparin. The HIT potential between bovine heparin and porcine heparin demonstrated no significant difference between the heparins (see MA responses in the image below). There were no lot to lot differences for the bovine heparins or the porcine heparins in either the platelet aggregation studies or the assessment for HIT. Conclusion In these studies of platelet function, the bovine and porcine source heparins were comparable with regards to their effects on platelet aggregation induced by multiple different agonists and their HIT potential. Figure. Figure. Disclosures Walenga: Eurofarma: Research Funding.