Annexin V Inhibition of Factor IXa-Catalyzed Factor X Activation on Human Platelets and on Negatively-Charged Phospholipid Vesicles

London, Fredda S.; Ahmad, Shafeeque; Walsh, Peter N.

doi:10.1021/bi960712v

Cited by 27 publications

(34 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…50 Likewise, annexin V is an incomplete inhibitor of the factor Xase complex on platelet membranes. 51 Our results, indicating that inhibition of the prothrombinase complex exceeds 80% at 60 nM annexin V only when the curvature of the membrane is minimal and the PS content is 15%, are in agreement with these prior studies. The ␤ 2 -glycoprotein I binds to PS-containing membranes and other negatively charged lipid-containing particles such as lipoproteins.…”

Section: Discussionsupporting

confidence: 93%

Lactadherin inhibits enzyme complexes of blood coagulation by competing for phospholipid-binding sites

Shi

Gilbert

2003

Blood

166

151

View full text Add to dashboard Cite

Lactadherin, a glycoprotein of the milk-fat globule membrane, contains tandem C domains with homology to discoidin-type lectins and to membrane-binding domains of blood-clotting factors V and VIII. We asked whether the structural homology confers the capacity to compete for the membrane-binding sites of factor VIII and factor V and to function as an anticoagulant. Our results indicate that lactadherin competes efficiently with factor VIII and factor V for binding sites on synthetic phosphatidylserine-containing membranes with half-maximal displacement at lactadherin concentrations of 1 to 4 nM. Binding competition correlated to functional inhibition of factor VIIIa-factor IXa (factor Xase) enzyme complex. In contrast to annexin V, lactadherin was an efficient inhibitor of the prothrombinase and the factor Xase complexes regardless of the degree of membrane curvature and the phosphatidylserine content. Lactadherin also inhibited the factor VIIa-tissue factor complex efficiently whereas annexin V was less effective. Because the inhibitory concentration of lactadherin was proportional to the phospholipid concentration, and because lactadherin was not an efficient inhibitor in the absence of phospholipid, the major inhibitory effect of lactadherin relates to blocking phospholipid sites rather than forming inhibitory protein-protein complexes. Lactadherin was also an effective inhibitor of a modified whole blood prothrombin time assay in which clotting was initiated by dilute tissue factor; 60 nM lactadherin prolonged the prothrombin time 150% IntroductionLactadherin is a 47 000-Da molecular weight (MW) glycoprotein of milk-fat globules. It has also been known as PAS-6/7, indicating the 2 glycosylation variants, 1 bovine-associated mucoprotein, BA-46, P47, and MFG-E8. 2 Lactadherin has a domain structure of EGF1-EGF2-C1-C2 in which EGF indicates epidermal growth factor homology domains, and the C domains share homology with the discoidin family, including the lipid-binding C domains of blood coagulation factor VIII and factor V. 2 The second EGF domain displays an Arg-Gly-Asp motif, 3 which binds to the ␣ v ␤ 3 and ␣ v ␤ 5 integrins. 1,[4][5][6] The second C domain binds to phospholipids. 6 In milk-fat globules, lactadherin lines the surface of the phospholipid bilayer that surrounds the central triglyceride droplet, apparently stabilizing the bilayer. 7 Lactadherin decreases the symptoms of rotavirus infection in infants, possibly by binding to rotavirus particles via carbohydrate moieties. 8 In tissue sections, lactadherin is found localized on the apical portion of secretory epithelium in the breast. 7 Abundant expression by breast carcinoma tissue makes lactadherin a potential target for antigen-guided radiation therapy. 9 Lactadherin is also found on the apical surface of epithelia in the biliary tree, the pancreas, and sweat glands 7 and is synthesized by aortic medial smooth muscle cells. 10 Function in these tissues remains unknown. Lactadherin has been identified as a zona pellucida-binding protein on the a...

show abstract

Section: Discussionsupporting

confidence: 93%

Lactadherin inhibits enzyme complexes of blood coagulation by competing for phospholipid-binding sites

Shi

Gilbert

2003

Blood

166

151

View full text Add to dashboard Cite

show abstract

“…The inhibition curves of FVIIIa in the presence of EGR-FIXa and FX are quite steep, and the slope of the curve is greater than that described by a simple competitive inhibition model. Thus, our equilibrium binding studies and competition studies further clarify that annexin V has an effect only on active cofactor (FVIIIa), similar to the effects we have reported on FIXa binding (53) and FX binding (23). We recently reported (53) on the kinetic effects of annexin V on FVIIIa enhancement of F-X activation on activated platelets and phospholipids.…”

Section: Discussionsupporting

confidence: 87%

“…Previously we have shown that annexin V is a very potent inhibitor of both FX and prothrombin binding to activated platelets with IC 50 of 3.1 and 2.6 nM, respectively (23). We have also shown that annexin V affects the affinity and stoichiometry of FIXa interaction with platelets and phospholipids, and we have concluded that the interaction of both the enzyme (FIXa) and the substrate (FX) is mediated in a complex manner by both phospholipids and perhaps a protein receptor (53). Studies from other laboratories (52,54) have reported that annexin V interferes with FV and FVa binding and prothrombinase activity on both the platelet membrane and phospholipid vesicles.…”

Section: Binding Of Pro-cofactor (Factor Viii) and Active Cofactor (Fmentioning

confidence: 70%

Structural and Functional Characterization of Platelet Receptor-mediated Factor VIII Binding

Ahmad¹,

Scandura²,

Walsh³

2000

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The importance of factor VIII (FVIII) 1 and FIX in hemostatic reactions is evident by the fact that hemophilia A (FVIII deficiency) and hemophilia B (FIX deficiency) are the two most serious congenital coagulation defects, both producing severe, life-threatening and life-long hemorrhagic disease. FVIII is synthesized as a single polypeptide chain containing 2,351 amino acids (1) and shows a discrete domain structure, A1-a1-A2-a2-B-a3-A3-C1-C2; the domains are separated by short spacers (a1, a2, and a3) composed of acidic regions that contain clusters of Asp and Glu residues (2). The A domains display ϳ30% homology to each other, and they also share sequence homology with ceruloplasmin and FV. The B domain is unique to FVIII. The C domains share homology with FV and discoidin 1, a lectin (1). During proteolytic activation of pro-cofactor (FVIII), the B domain that does not contribute to cofactor function is excised. The heavy chain (HC) and light chain (LC) remain noncovalently associated through the A1 and A3 domains in a metal ion-dependent manner and participate as an active cofactor in tenase complex formation (1). The following terminology, suggested by Lenting et al. (2), for FVIII subunits was used: A1 ϭ HC-derived fragment containing residues 1-372; A2 ϭ HC-derived fragment containing residues 373-740; A1-A2 ϭ HC subunit of FVIII containing residues 1-740 (without the B domain); B ϭ HC subunit residues 741-1648; LC ϭ LC of FVIII; A3-C1-C2 ϭ LC-derived fragment arising from the cleavage at position 1689. Thrombin cleaves FVIII at one specific site within the LC, Arg 1689 , and at two sites in the HC, Arg 372 and Arg 750 , resulting in a heterodimer of 50 kDa (A1 domain), 43 kDa (A2 domain), and 73 kDa (A3-C1-C2 dimer); all of these subunits are required for the procoagulant activity (3). More specifically, the cleavages at Arg 372 and Arg 1689 are required to exert full cofactor activity (4, 5), whereas cleavage of the LC at Arg 1689 is also responsible for dissociation of FVIII from von Willebrand factor (vWF) (6).FVIII in the circulation forms a tight, noncovalent complex with vWF, and both the amino-terminal and the carboxylterminal regions of FVIII are involved in this interaction (6, 7). The formation of the FVIII-vWF complex is essential for the survival of FVIII in the circulation. Another functional consequence of FVIII-vWF complex formation is to prevent binding of FVIII to components of the FX-activating complex, e.g. the binding of FVIII LC to FIXa has been shown to be inhibited by vWF (8). Furthermore, FVIII-vWF complex is less susceptible to proteolytic degradation by proteases like activated protein C (9) and FXa (10). vWF also prevents FVIII binding to phospholipids (11) and platelets (12). In contrast, vWF does not protect FVIII from thrombin cleavage (13,14). Recently, it has been shown (15) that FIXa displays similar affinity for pro-cofactor (FVIII) and active cofactor (FVIIIa), but as mentioned above, the FIXa-binding site is not accessible when FVIII is in com-

show abstract

“…FVIII (200 U/ ml) was incubated 1 minute at 37°C with thrombin (0.5 U/ml) and an additional minute with hirudin (5 U/ml) immediately before addition to reaction mixtures. Reactions were stopped after 2-3 minutes and were analyzed for FXa generated by chromogenic substrate S-2765 hydrolysis as described (47). Negative controls included reactions missing the enzyme, reactions missing platelets, and reactions using unstimulated platelets.…”

Section: Fxa Generationmentioning

confidence: 99%

PAR-1-Stimulated Factor IXa Binding to a Small Platelet Subpopulation Requires a Pronounced and Sustained Increase of Cytoplasmic Calcium

2006

Self Cite

View full text Add to dashboard Cite

We previously reported that only a subpopulation of PAR-1-stimulated platelets binds coagulation factor IXa, since confirmed by other laboratories. Since calcium changes have been implicated in exposure of procoagulant aminophospholipids, we have now examined calcium fluxes in this subpopulation by measuring fluorescence changes in Fura Red/AM-loaded platelets following PAR-1 stimulation. While fluorescence changes in all platelets indicated calcium release from internal stores and influx of external calcium, a subpopulation of platelets displayed a pronounced increase in calcium transients by 15 seconds and positive factor IXa binding by 2 minutes, with calcium transients sustained for 45 minutes. Pretreatment of platelets with Xestospongin C to inhibit IP3-mediated dense tubule calcium release, and the presence of impermeable calcium channel blockers nifedipine, SKF96365 or LaCl 3, inhibited PAR-1-induced development of a subpopulation with pronounced calcium transients, factor IXa binding, and platelet support of FXa generation, suggesting the importance of both release of calcium from internal stores and influx of extracellular calcium. When platelets were stimulated in EDTA for 5 to 20 minutes before addition of calcium, factor IXa binding sites developed on a smaller subpopulation but with unchanged rate indicating sustained opening of calcium channels and continued availability of signaling elements required for binding site exposure. While pretreatment of platelets with 100 μM BAPTA/AM (K d 160 nM) had minimal effects, 100 μM 5, 5′-dimethylBAPTA/AM (K d 40 nM) completely inhibited the appearance and function of the platelet subpopulation, indicating the importance of minor increases of cytoplasmic calcium. We conclude that PAR-1-stimulated development of factor IXa binding sites in a subpopulation of platelets is dependent upon release of calcium from internal stores leading to sustained and pronounced calcium transients.An essential event in the hemostatic response to vascular injury is the assembly of the factor X (FX) 1 activating complex on the surface of activated platelets (1,2). The assembly of this important enzymatic complex requires the exposure of coagulation protein binding sites on the surface of platelets activated with thrombin or collagen but not with adenosine diphosphate (3-6). All proteins required for physiologically relevant platelet-supported FX-activation, including the enzyme factor IXa (FIXa), the cofactor factor VIIIa (FVIIIa) and the substrate factor X (FX), must be bound to their respective receptors on activated platelets (7). Thus, the zymogen FIX binds with high affinity (K d ~2.5 nM) to a discreet number of platelet receptors † This study was supported by research grants from the National Institute of Health: HL70683, HL46213 and HL74124. *To whom correspondence should be addressed: Fredda London, Ph.D., Sol Sherry Thrombosis Research Center, Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140, Tel: 215-707-4458; Fax: 215-707-3005;...

show abstract

Annexin V Inhibition of Factor IXa-Catalyzed Factor X Activation on Human Platelets and on Negatively-Charged Phospholipid Vesicles

Cited by 27 publications

References 27 publications

Lactadherin inhibits enzyme complexes of blood coagulation by competing for phospholipid-binding sites

Lactadherin inhibits enzyme complexes of blood coagulation by competing for phospholipid-binding sites

Structural and Functional Characterization of Platelet Receptor-mediated Factor VIII Binding

PAR-1-Stimulated Factor IXa Binding to a Small Platelet Subpopulation Requires a Pronounced and Sustained Increase of Cytoplasmic Calcium

Contact Info

Product

Resources

About