Platelet GPIIbIIIa enriched membrane protrusions (Tether) in healthy and diseased individuals

Introduction Platelets mediate adhesion and aggregation via membrane glycoproteins. The glycoprotein (GP)IIbIIIa binds to fibrinogen and other matrix proteins. Recently we observed platelets, forming long, thin membrane protrusions (tethers) that were enriched with GPIIbIIIa in some patients ([Fig.1]). These tethers are very similar to Platelet-derived Integrin & Tetraspanin-rich Tethers (PITTs) described in mouse models [1]. The clinical implications of these tethers remain unclear. We assessed the frequency of this finding in patients with myeloproliferative neoplasms (MPN). MPN are associated with an increased risk for thrombotic complications.

Fig. 1 Pattern of the index-patient; The patient presented with intense formation of GPIIbIIIa-enriched membrane protrusions (A) and reduced alpha granule content as represented by staining of vWf(B) and P-selectin (C).

Method We analyzed blood smears of patients with confirmed MPN (n=135); healthy blood donors ( n=32) served as control. Blood smears were stained with 13 primary antibodies targeting platelet structures, e.g. anti CD41 (IIbIIIa), IbIX, CD62P, CD63, von Willebrand factor (vWf). After labeling with fluorescent secondary antibodies, platelets were assessed by immunofluorescence microscopy. At least 5 fields of views have been assessed for each blood smear. Parameters such as length and frequency of tethers were documented.

Results Tethers were observed in 48/135 (36%) MPN patients. Tethers stained only for GPIIbIIIa, but not for GPIbIX [2]. In about half of tether-positive patients (24/48), tethers were short and present in less than 30% of the assessed platelet population. Only six patients presented long tethers in a majority of the assessed platelets. The remaining 18 patients displayed moderate tether formation. Furthermore, 32/48 (67%) tether-positive patients had diminished content of α-granules and 19 of them had a combined reduction of α- and δ-granules. Within the cohort of all 135 MPN patients, 46 (34%) presented with an arterial or venous thrombotic event in their history; for 14 patients this clinical information was not available. Of the 46 MPN patients with thrombotic events 14 (30%) formed tethers compared to 30 (39%) of the subgroup without thrombotic events. In our healthy control cohort 2/32 (6%) individuals presented with short tethers in less than 10% of platelets ([Fig. 2]). Both individuals were female, under the age of 35 years and without any documented chronic disease or medication intake.

Fig. 2 Screening of healthy individuals and patients with MPN; a cohort of healthy individuals (n=32) and a cohort of patients with MPN (n=135) have been assessed for platelet tethers. Tethers were more commonly found in patients with MPN and often associated with alterations of platelet granule content. Thrombotic events occurred in 34 % of the patients; 30 % of the subpopulation with thrombotic events displayed tethers.

Conclusion Platelet tethers expressing GPIIbIIIa are present in a subgroup of MPN patients but without strong association to thrombosis. Long thin membrane protrusions enriched with GPIIbIIIa are usually not found in the normal population, while short pseudopodia-like protrusions are also seen in about 5-10% of healthy subjects. Besides the formation of tether, we noticed further alterations of platelet morphology such as reduced α-and δ-granule content in the patient cohort. This suggests an in-vivo activation of the platelets. Further patient populations with prothrombotic disorders may need to be assessed to better understand the biological role of tethers/PITTs in humans.

Publikationsverlauf

Artikel online veröffentlicht:
20. Februar 2023

• References

• 1 Kusch C, Stegner D, Johnson D. et al novel and unique mechanism of αIIbβ3 regulation in platelets.[Abstract] ISTH congress 2022 https://abstracts.isth.org/abstract/a-novel-and-unique-mechanism-of-%ce%b1iib%ce%b23-regulation-in-platelets
• 2 Dopheide SM, Maxwell MJ, Jackson SP. Shear-dependent tether formation during platelet translocation on von Willebrand factor. [Article] Blood 2002; 99 (01) 159-67