Introduction: Antiphospholipid syndrome (APS) is defined by thrombosis or fetal loss in the presence of persistent circulating antiphospholipid antibodies (aPL). Several studies have identified activated circulating platelets in APS. Our recent work demonstrates that patients with APS have increased levels of circulating “procoagulant” platelets defined by surface expression of phosphatidylserine (PS) and P-selectin (Psel) that correlate with platelet bound C4d. This suggests a role for complement in stimulating PS expression. To better define the significance of platelet PS expression in APS and the platelet phenotype contribution to the procoagulant state, we measured PS and Psel expression on freshly isolated platelets from APS patients in parallel with the platelets' procoagulant activity.

Methods: Washed platelets from APS patients and healthy donors (HD) were isolated from whole blood by differential centrifugation and analyzed for PS and Psel expression by flow cytometry using annexin V and anti-Psel antibodies. Platelets were considered PS+ if expression was more than 2 standard deviations higher than HD platelets. For calibrated automated thrombinography (CAT), platelets were resuspended in pooled HD plasma to a count of 0.5 x 108 cells/mL. Platelet-containing plasma samples were split into 2 groups of triplicates, with 1 triplicate treated with a thrombin calibrator while the other was treated with a PRP-specific reagent containing tissue factor and minimal phospholipid. After incubation, a mixture of CaCl2 and fluorogenic substrate was simultaneously added to all sample wells. Fluorescence was read over 1 hour and analyzed by thrombinoscope software. Kinetic parameters of interest were thrombin generation rate (velocity index), maximum thrombin concentration (peak thrombin), and time to maximum thrombin concentration (time to peak).

Results: We assessed platelet samples from 7 patients with APS (3 female, average age 52.0 (range: 29.2-62.2) years) and 3 HD. Three distinct groups were compared in the analysis of thrombin generation: APS patients with PS exposing platelets (PS+), APS patients without PS exposing platelets (PS-), and HD. Five of the 7 APS patients had PS+ platelets. Of the patients with PS+ platelets, 4 were triple positive for aPL (LAC+, aCL and B2GP1 IgG or IgM >150) with a history of VTE and 1 had low-positive aPL (LAC+, low aCL and B2GP1 IgG, nondetectable IgM) with VTE. All but 1 of the PS+ patients were on anticoagulation, and 2 were on aspirin. Of the 2 APS patients with PS- platelets, 1 had strong positive aPL and a history of VTE, and the other had low-positive aPL with VTE. Both PS- patients were on anticoagulation, and neither on aspirin.

Peak thrombin generation was significantly higher in the presence of PS+ platelets than HD platelets (PS+: 88.7 [95% CI: 76.1-101.3] vs HD: 56.9 [95% CI: 49.4-64.5], P = 0.001). Peak thrombin generation supported by PS+ versus PS- platelets was not significantly different but trended towards an increase (PS+: 88.7 [95% CI: 76.1-101.3] vs PS-: 71.4 [95% CI: 60.1-82.8], P = 0.15). Time to peak thrombin generation was significantly decreased in PS+ platelets compared to PS- APS platelets and HD platelets (PS+: 17.0 [95% CI: 15.8-18.2], PS-: 20.1 [95% CI: 18.2-22.0], P = 0.01; HD: 21.6 [95% CI: 19.6-23.7], P = <0.001). The velocity index in PS+ platelets was significantly increased compared to PS- APS and HD platelets (PS+: 9.6 [95% CI: 7.8-11.5], PS- 5.7 [95% CI: 4.9-6.6], P = 0.02; HD: 4.6 [95% CI: 3.2-6.0], P = <0.001).

No significant differences were observed between PS- APS platelets and HD with respect to peak thrombin (P = 0.73), time to peak (P = 0.42), or velocity index (P = 0.39).

Conclusions: These studies, in which platelets from APS patients were suspended in pooled HD plasma and used as a phospholipid source for thrombin generation, enable study of the platelet contribution to thrombin generation in patients on anticoagulation therapy. PS+ platelets from APS patients support more rapid and total thrombin generation compared to platelets from HD or APS patients whose platelets do not express PS. We suggest that PS+ platelets contribute to the procoagulant state in at least some APS patients. The study is limited by the small sample size and heterogeneity of APS patients. Further studies assessing thrombin generation supported by PS+ platelets from patients with APS may be useful in thrombotic risk stratification.

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2025
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