Background: Transgenic mice that ectopically express uPA in MKs have a mild bleeding diathesis, but no systemic fibrinolysis. Infusion of uPA-containing platelets into wildtype mice prevented thrombus formation in a carotid artery-injury model.

Aims: We wish to develop a clinically relevant model to deliver such anti-thrombotic platelets, taking advantage of advances in in vitro MK generation and their progeny platelets by endogenous uPA expression or MK uptake of exogenous uPA. As noted above, infused platelets endogenously expressing scuPA prevented carotid artery thrombus formation. We now tested whether platelets derived from scuPA-loaded CD34⁺-MKs would similarly prevent thrombus formation.

Methods: We used a carotid injury model in NOD-scid IL2rγnull (NSG) mice homozygous for VWF^R1326H (a mutation switching binding VWF specificity from mouse to human GPIb/IX). NSG/VWF^R1326H mice  had impaired clotting after vascular injury compared to NSG mice unless infused with human platelets or MKs (Figure 2). 

Results: We observed that CD34⁺ hematopoietic cell-derived MKs internalize and store single-chain uPA (scuPA) and a truncated uPA mutant, modified to be plasmin-resistant but thrombin activatable (uPA-T), in identical α-granules (Figure 1A), but in completely different granules from endogenously expressed scuPA following lentiviral transduction (Figure 1B). Endocytosed uPA showed little-to-no co-localization with endogenous von Willebrand Factor (VWF) and platelet factor 4, but significant colocalization with endocytosed Factor V. Uptake of Factor V and uPA were both inhibited by the LRP1 antagonist receptor-associated protein (RAP) and anti-LRP1 antibodies.Significantly less post-injury clotting was seen upon infusion of scuPA-loaded MKs (Figure 2). These data show that MKs internalize potentially clinically relevant concentrations of uPA through a process likely to involve LRP1, ending up in a distinct, exogenous-uptake α-granule pool.

Uptake and storage of scuPA, uPA-T and ectopically expressed uPA by CD34+ MKs.

Preventing nascent thrombi by endocytosed scuPA in a Rose Bengal, carotid artery photochemical injury murine model

Conclusions: Future studies will expand on understanding these distinct α-granule pool and whether endogenous or exogenous uPA in in vitro-grown MKs can be a point-of-care thromboprophylaxis in the setting of high-risk.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/defining-two-separate-but-functional-%ce%b1-granule-pools-in-in-vitro-grown-megakaryocytes-mks-lessons-from-urokinase-plasminogen-activator-upa/