Kinetic regulation of the binding of prothrombin to phospholipid membranes

Smith, Emma C.; Vekaria, Rina; Brown, Katherine A.; Longstaff, Colin

doi:10.1007/s11010-013-1735-2

Cited by 8 publications

(7 citation statements)

References 38 publications

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“…The ability of the FLAG‐tagged rCEA N module to bind directly to immobilized Fn, rCEA N and A 3 domains, in a dose dependent manner led to the derivation (Figures 2, 3 and 6) of dissociation constants from ELISA‐based binding curves (Figure 7) using established protocols (Sato et al., 2009; Smith et al., 2013; Jardim et al., 2000). The calculated ratios of bound to free FLAG‐tagged rCEA N module with individual monomeric components revealed that N‐to‐N, N‐to‐A 3 and N‐to‐Fn interactions to be of moderate to high affinity with K D values of 100 ± 17 nM, 18 ± 3 nM and 16 ± 3 nM for the binding of FLAG‐tagged rCEA N to immobilized rCEA N, rCEA A 3 and Fn, respectively.…”

Section: Resultsmentioning

confidence: 99%

“…Equilibrium dissociation constants for the CEA N domain interaction with itself, the A 3 domain and with Fn were derived by ELISA as previously described (Sato et al., 2009; Smith et al., 2013; Jardim et al., 2000). Briefly, increasing concentrations of FLAG‐tagged rCEA N were added to multi‐well plates coated with 1 μg of either Fn, rCEA N or rCEA A 3 domains.…”

Section: Methodsmentioning

confidence: 99%

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The carcinoembryonic antigen IgV‐like N domain plays a critical role in the implantation of metastatic tumor cells

et al. 2013

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The human carcinoembryonic antigen (CEA) is a cell adhesion molecule involved in both homotypic and heterotypic interactions. The aberrant overexpression of CEA on adenocarcinoma cells correlates with their increased metastatic potential. Yet, the mechanism(s) by which its adhesive properties can lead to the implantation of circulating tumor cells and expansion of metastatic foci remains to be established. In this study, we demonstrate that the IgV-like N terminal domain of CEA directly participates in the implantation of cancer cells through its homotypic and heterotypic binding properties. Specifically, we determined that the recombinant N terminal domain of CEA directly binds to fibronectin (Fn) with a dissociation constant in the nanomolar range (K(D) 16 ± 3 nM) and interacts with itself (K(D) 100 ± 17 nM) and more tightly to the IgC-like A(3) domain (K(D) 18 ± 3 nM). Disruption of these molecular associations through the addition of antibodies specific to the CEA N or A(3)B(3) domains, or by adding soluble recombinant forms of the CEA N, A(3) or A(3)B(3) domains or a peptide corresponding to residues 108-115 of CEA resulted in the inhibition of CEA-mediated intercellular aggregation and adherence events in vitro. Finally, pretreating CEA-expressing murine colonic carcinoma cells (MC38.CEA) with rCEA N, A3 or A(3)B(3) modules blocked their implantation and the establishment of tumor foci in vivo. Together, these results suggest a new mechanistic insight into how the CEA IgV-like N domain participates in cellular events that can have a macroscopic impact in terms of cancer progression and metastasis.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The carcinoembryonic antigen IgV‐like N domain plays a critical role in the implantation of metastatic tumor cells

et al. 2013

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“…There are nine conserved γ-carboxyglutamic acid residues, and the additional γ-carboxyglutamic acid at position 32 has been shown to be dispensable for prothrombin activation by the prothrombinase complex yet required for binding to phospholipids (32). Residue 32 has been implied for high-affinity binding and interspecies differences among naturally occurring vitamin K-dependent proteins S, Z, and C (33) and explains the peculiar kinetics of prothrombin binding to membranes (34). The Gla domain of ProTΔ146-167 and its linker connection to kringle-1 are clearly detected from A1 to N64 with four helices spanning residues N12-T21 (helix 1), S23-L31 (helix 2), T37-A50 (helix 3), P53-G63 (helix 4), and the hydrophobic ω-loop (F4-L5-V8) primed for membrane binding.…”

Section: Activationmentioning

confidence: 99%

The linker connecting the two kringles plays a key role in prothrombin activation

Pozzi

Chen

Pelc

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

100

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The zymogen prothrombin is proteolytically converted by factor Xa to the active protease thrombin in a reaction that is accelerated >3,000-fold by cofactor Va. This physiologically important effect is paradigmatic of analogous cofactor-dependent reactions in the coagulation and complement cascades, but its structural determinants remain poorly understood. Prothrombin has three linkers connecting the N-terminal Gla domain to kringle-1 (Lnk1), the two kringles (Lnk2), and kringle-2 to the C-terminal protease domain (Lnk3). Recent developments indicate that the linkers, and particularly Lnk2, confer on the zymogen significant flexibility in solution and enable prothrombin to sample alternative conformations. The role of this flexibility in the context of prothrombin activation was tested with several deletions. Removal of Lnk2 in almost its entirety (ProTΔ146-167) drastically reduces the enhancement of thrombin generation by cofactor Va from >3,000-fold to 60-fold because of a significant increase in the rate of activation in the absence of cofactor. Deletion of Lnk2 mimics the action of cofactor Va and offers insights into how prothrombin is activated at the molecular level. The crystal structure of ProTΔ146-167 reveals a contorted architecture where the domains are not vertically stacked, kringle-1 comes within 9 Å of the protease domain, and the Gladomain primed for membrane binding comes in contact with kringle-2. These findings broaden our molecular understanding of a key reaction of the blood coagulation cascade where cofactor Va enhances activation of prothrombin by factor Xa by compressing Lnk2 and morphing prothrombin into a conformation similar to the structure of ProTΔ146-167.clotting factor | zymogen activation | structural biology T he clotting factor prothrombin is proteolytically converted by factor Xa to the active protease thrombin by cleavage at two sites, R271 and R320, along two mutually exclusive pathways producing the intermediates prethrombin-2 (cleavage at R271 first) or meizothrombin (cleavage at R320 first). The reaction is greatly accelerated through a drastic increase in k cat when catalyzed by the prothrombinase complex composed of factor Xa, cofactor Va, Ca 2+ , and phospholipids (1, 2). Both the rate enhancement and pathway of activation are under the control of cofactor Va and phospholipids (3, 4), although the cofactor has the greatest effect in enhancing k cat . The molecular determinants of this physiologically important effect are of substantial mechanistic interest because they bear on the cofactor-dependent activation of zymogens in many proteolytic cascades (5, 6).Prothrombin is composed of fragment 1 (residues 1-155), fragment 2 (residues 156-271), and a protease domain (residues 272-579). Fragment 1 contains the Gla domain (residues 1-46) and kringle-1 (residues 65-143), fragment 2 contains kringle-2 (residues 170-248), and the protease domain contains the A chain (residues 272-320) and the catalytic B chain (residues 321-579). Three linkers-to be referred to as Lnk1, Lnk2, ...

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“…It can be a result of high cross-reactivity between platelet membrane and PARs for binding with MT. Affinity of FII to PL (Kd = 20 -160 nM depending on membrane composition [28]- [30]) is comparable with affinity of FIIa to PARs (Kd = 11 -16 nM and 2.9 -4.6 μM depending on receptor type [31] [32]). But FIIa binds to PAR by both active site and exosite I [23] [33].…”

Section: Discussionmentioning

confidence: 99%

Meizothrombin Preparation and Its Role in Fibrin Formation and Platelet Aggregation

Korolova¹,

Chernyshenko²,

Gornytska³

et al. 2014

ABB

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Meizothrombin (MT) is one of prothrombin derivatives which appears in haemostasis activation area. However, its role in haemostasis regulation isn't clear. We studied the role of MT in fibrin formation, platelet activation and aggregation. A new effective method of obtaining MT from native human prothrombin was developed using immobilised prothrombin activator from Echis multisquamatis venom. The protein was stable and electrophoretically pure. Platelet-rich plasma for aggregation study and gel-sieved platelets for flow-cytometry were separated from blood of healthy donors. It was shown that MT transformed fibrinogen to fibrin and activated clotting factor XIII. MT didn't activate gel-sieved intact platelets, but in platelet-rich plasma, increased platelet aggregation induced by ADP, collagen and adrenalin.

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Kinetic regulation of the binding of prothrombin to phospholipid membranes

Cited by 8 publications

References 38 publications

The carcinoembryonic antigen IgV‐like N domain plays a critical role in the implantation of metastatic tumor cells

The carcinoembryonic antigen IgV‐like N domain plays a critical role in the implantation of metastatic tumor cells

The linker connecting the two kringles plays a key role in prothrombin activation

Meizothrombin Preparation and Its Role in Fibrin Formation and Platelet Aggregation

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