Packaging of supplemented urokinase into naked alpha-granules of<i>in vitro</i>-grown megakaryocytes for targeted therapeutic delivery
Abstract Our prior finding that uPA endogenously expressed and stored in the platelets of transgenic mice prevented thrombus formation without causing bleeding, prompted us to develop a potentially clinically relevant means of generating anti-thrombotic human plateletsin vitrofrom CD34+hematopoietic cell-derived megakaryocytes. CD34+-megakaryocytes internalize and store in α-granules single-chain uPA (scuPA) and a uPA variant modified to be plasmin-resistant, but thrombin-activatable, (uPAT). Both uPAs co-localized with internalized factor V (FV), fibrinogen and plasminogen, low-density lipoprotein receptor-related protein 1 (LRP1), and interferon-induced transmembrane protein 3 (IFITM3), but not with endogenous von Willebrand factor (VWF). Endocytosis of uPA by CD34+-\megakaryocytes was mediated in part via LRP1 and αIIbβ3. scuPA-containing megakaryocytes degraded endocytosed intragranular FV, but not endogenous VWF, in the presence of internalized plasminogen, whereas uPAT-megakaryocytes did not significantly degrade either protein. We used a carotid-artery injury model in NOD-scid IL2rγnull (NSG) mice homozygous for VWFR1326H(a mutation switching binding VWF specificity from mouse to human glycoprotein IbmlIX) to test whether platelets derived from scuPA-MKs or uPAT-Mks would prevent thrombus formation. NSG/VWFR1326Hmice exhibited a lower thrombotic burden after carotid artery injury compared to NSG mice unless infused with human platelets or MKs, whereas intravenous injection of either uPA-containing megakaryocytes into NSG/VWFR1326Hgenerated sufficient uPA-containing human platelets to lyse nascent thrombi. These studies suggest the potential to deliver uPA or potentially other ectopic proteins within platelet α-granules fromin vitro-generated megakaryocytes.Key points <jats:list list-type="bullet">Unlike platelets, in vitro-grown megakaryocytes can store exogenous uPA in its α-granules.uPA uptake involves LRP1 and αIIbβ3 receptors and is functionally available from activated platelets..
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Preprint| Erscheinungsjahr: |
2023 |
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| Erschienen: |
2023 |
| Enthalten in: |
bioRxiv.org - (2023) vom: 12. Dez. Zur Gesamtaufnahme - year:2023 |
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| Sprache: |
Englisch |
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| Beteiligte Personen: |
Poncz, Mortimer [VerfasserIn] |
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| Links: |
Volltext [kostenfrei] |
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| doi: |
10.1101/2023.12.05.570278 |
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| PPN (Katalog-ID): |
XBI041780809 |
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| 100 | 1 | |a Poncz, Mortimer |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Packaging of supplemented urokinase into naked alpha-granules of<i>in vitro</i>-grown megakaryocytes for targeted therapeutic delivery |
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| 520 | |a Abstract Our prior finding that uPA endogenously expressed and stored in the platelets of transgenic mice prevented thrombus formation without causing bleeding, prompted us to develop a potentially clinically relevant means of generating anti-thrombotic human plateletsin vitrofrom CD34+hematopoietic cell-derived megakaryocytes. CD34+-megakaryocytes internalize and store in α-granules single-chain uPA (scuPA) and a uPA variant modified to be plasmin-resistant, but thrombin-activatable, (uPAT). Both uPAs co-localized with internalized factor V (FV), fibrinogen and plasminogen, low-density lipoprotein receptor-related protein 1 (LRP1), and interferon-induced transmembrane protein 3 (IFITM3), but not with endogenous von Willebrand factor (VWF). Endocytosis of uPA by CD34+-\megakaryocytes was mediated in part via LRP1 and αIIbβ3. scuPA-containing megakaryocytes degraded endocytosed intragranular FV, but not endogenous VWF, in the presence of internalized plasminogen, whereas uPAT-megakaryocytes did not significantly degrade either protein. We used a carotid-artery injury model in NOD-scid IL2rγnull (NSG) mice homozygous for VWFR1326H(a mutation switching binding VWF specificity from mouse to human glycoprotein IbmlIX) to test whether platelets derived from scuPA-MKs or uPAT-Mks would prevent thrombus formation. NSG/VWFR1326Hmice exhibited a lower thrombotic burden after carotid artery injury compared to NSG mice unless infused with human platelets or MKs, whereas intravenous injection of either uPA-containing megakaryocytes into NSG/VWFR1326Hgenerated sufficient uPA-containing human platelets to lyse nascent thrombi. These studies suggest the potential to deliver uPA or potentially other ectopic proteins within platelet α-granules fromin vitro-generated megakaryocytes.Key points <jats:list list-type="bullet">Unlike platelets, in vitro-grown megakaryocytes can store exogenous uPA in its α-granules.uPA uptake involves LRP1 and αIIbβ3 receptors and is functionally available from activated platelets. | ||
| 650 | 4 | |a Biology |7 (dpeaa)DE-84 | |
| 650 | 4 | |a 570 |7 (dpeaa)DE-84 | |
| 700 | 1 | |a Zaitsev, Sergei V. |4 aut | |
| 700 | 1 | |a Ahn, Hyunsook |4 aut | |
| 700 | 1 | |a Kowalska, M. Anna |4 aut | |
| 700 | 1 | |a Bdeir, Khalil |4 aut | |
| 700 | 1 | |a Camire, Rodney M. |4 aut | |
| 700 | 1 | |a Cines, Douglas B. |4 aut | |
| 700 | 1 | |a Stepanova, Victoria |0 (orcid)0000-0002-9313-5742 |4 aut | |
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