Regulation of factor V by the anticoagulant protease activated protein C: Influence of the B-domain and TFPIα

Ayombil, Francis; Petrillo, Teodolinda; Kim, Haein; Camire, Rodney M.

doi:10.1016/j.jbc.2022.102558

Cited by 6 publications

(4 citation statements)

References 56 publications

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“…In this position, TFPIα would also encroach on R506 ( Figure 2 AB), thereby explaining the difficulty of interaction with fV Leiden (R506Q) 54 , 55 and the ability to protect R506 from APC cleavage. 27 Similar structural interactions are expected of the BR in fV and account for how intramolecular binding to the AR may keep the cofactor in its inactive state. 1 , 5 , 6 , 7 , 9 , 53 The third Kunitz domain engages PS likely bound to HC1, HC2, and HC4 of the AR ( Figure 6 ) and in contact with fXa, bound along the A2, A3, C1 domains as observed in the prothrombinase complex 16 (see Figure 5 ) and inhibited by the second Kunitz domain.…”

Section: Discussionmentioning

confidence: 81%

“… 7 The proximity to R506 also explains the TFPIα difficult interaction with fV Leiden (R506Q) 54 , 55 and its ability to protect R506 from APC cleavage. 27 A backbone clash (orange oval) involves the AR segment 1530 INSSRDPDNIAAVYLR 1545 of fV short with the segment 86 RKLCSLDN 93 of the EGF2 domain of fXa. Removal of this clash would require a modest (<3 Å) relative rearrangement of the 2 segments.…”

Section: Resultsmentioning

confidence: 99%

“…Elucidation of the architecture of the B domain also bears significant clinical relevance. 26 , 27 , 28 , 29 , 30 , 31 , 32 The East Texas bleeding disorder is associated with a splice fV isoform, called fV short, resulting in the deletion of residues from 756 to 1458 that include the BR and 31 tandemly arranged repeats of the B domain 4 , 31 , 32 ( Figure 1 B). Loss of the BR unmasks a high-affinity binding site in the AR for TFPIα, a key regulator of the assembly of prothrombinase 29 , 30 and blood coagulation.…”

Section: Introductionmentioning

confidence: 99%

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Cryo-EM structure of coagulation factor V short

Mohammed

Pelc

Rau

et al. 2023

Blood

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Coagulation factor V (fV) is the inactive precursor of fVa, an essential component of the prothrombinase complex required for rapid activation of prothrombin in the penultimate step of the coagulation cascade. In addition, fV regulates the tissue factor pathway inhibitor α (TFPIα) and protein C pathways that inhibit the coagulation response. A recent cryogenic electron microscopy (cryo-EM) structure of fV has revealed the architecture of its A1-A2-B-A3-C1-C2 assembly but left the mechanism that keeps fV in its inactive state unresolved due to intrinsic disorder in the B domain. A splice variant of fV, fV short, carries a large deletion of the B domain that produces constitutive fVa-like activity and unmasks epitopes for binding of TFPIα. The cryo-EM structure of fV short was solved at 3.2 Å resolution and reveals the arrangement of the entire A1-A2-B-A3-C1-C2 assembly for the first time. The shorter B domain stretches across the entire width of the protein, making contacts with the A1, A2 and A3 domains but suspended over the C1 and C2 domains. In the portion distal to the splice site, several hydrophobic clusters and acidic residues provide a likely binding site for the basic C-terminal end of TFPIα. In fV, these epitopes may bind intramolecularly the basic region of the B domain. The cryo-EM structure reported in this study advances our understanding of the mechanism that keeps fV in its inactive state, provides new targets for mutagenesis and facilitates future structural analysis of fV short in complex with TFPIα, protein S and fXa.

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Section: Discussionmentioning

confidence: 81%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Cryo-EM structure of coagulation factor V short

Mohammed

Pelc

Rau

et al. 2023

Blood

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“…Western blotting of -granule proteins CD34 + -MKs differentiated for 10 days were incubated with purified plasmin-free plasminogen at 20 µg/mL for 24 hours, washed and then incubated for an additional 18 hours with recombinant human scuPA or uPAT (200 nM each) 11 in the absence or presence of human FV (400 nM) 29 . The cells were then lysed in 1×radioimmunoprecipitation assay (RIPA) buffer with added 1× proteinase inhibitor mixture (Sigma) and 1× phosphatase inhibitors mixture 2 (Sigma).…”

Section: Generation Of Upa-containing Megakaryocytes (Upa-mks) and Fl...mentioning

confidence: 99%

Packaging of supplemented urokinase into naked alpha-granules ofin vitro-grown megakaryocytes for targeted therapeutic delivery

Poncz,

Zaitsev,

Ahn

et al. 2023

Preprint

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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 pointsUnlike 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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