Medical Hypotheses 84 (2015) 434–436

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Attenuating a sickle cell crisis with annexin V James Randall Kennedy ⇑ Manatee Memorial Hospital, Bradenton, FL, USA

a r t i c l e

i n f o

Article history: Received 17 October 2014 Accepted 23 January 2015

a b s t r a c t A sickle cell crisis is a painful and dangerous condition that defies effective treatment but fortunately it usually terminates spontaneously and patients spend far more time crisis free than in its painful throes. This suggests that an unstable physiologic balance exists between steady state sickle cell disease (SCD) and the crisis state and if this is so a therapeutic nudge during a crisis may help to terminate it. Annexin V may be able to provide this push. The phosphatidylserine (PS) molecules normally appear on the surface of senescent erythrocytes where they are recognized by macrophages and rapidly removed so that normally only about 1% are present in the circulation but in SCD 30–40% are prematurely senescent and their removal is delayed. The PS+ sickle erythrocytes remaining in the circulation adhere to the endothelium and their exposed PS acts as a platform for the initiation of the coagulation cascade that is responsible for clot propagation. Annexin V’s great affinity for PS allows it to bond to it forming a shield that blocks both of these actions suggesting that its therapeutic administration during a sickle crisis may be able to hasten its termination. Ó 2015 The Author. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction The treatment of a sickle cell crisis has been and still is support and comfort measures and though this has significantly improved over the years we are still unable to shorten its course. The transitory nature of a crisis suggests that a tenuous balance exists between it and the steady state suggesting that even a slight assist to its natural physiologic correction could hasten its end. The adhesive and procoagulant PS molecules present on the surface of prematurely senescent sickle erythrocytes provides an avenue for such an assist and the annexin V molecule provides the means. This paper will address the normal physiologic functions of PS and annexin V and attempt to explain the circumstances present in SCD that causes PS display on membrane surfaces to become pathologic and how annexin may be able to prevent this. The hypothesis The hypothesis presented is based on the fact that approximately 1/3 of sickle erythrocytes are prematurely senescent causing them to display PS molecules on their surface and that the adhesive and procoagulant character of this molecule is Abbreviations: PS, phosphatidylserine; SCD, sickle cell disease; TBS, thrombospondin. ⇑ Address: 4704 Riverview Blvd, Bradenton, FL 34209, USA. Tel.: +1 941 746 1208. E-mail address: [email protected]

responsible for a major portion of these two pathologic components in SCD. In vitro studies show that the annexin V molecule blocks approximately 1/3 of the endothelial adhesion in SCD and it also blocks initiation of the blood factor coagulation cascade. Annexin V levels are naturally elevated in SCD and it is proposed that supra physiologic amounts of it administered during a crisis may be able to hasten its termination.

Phosphatidylserine As erythrocytes become senescent they begin to display molecules on their surface that label them for elimination and one of them is PS. The PS molecule is normally on the inner leaflet of the cell membrane but it is displayed on the surface of senescent, dysfunctional or defective cells when they self-destruct by apoptotic fragmentation of the cell nucleus. However, erythrocytes have no nucleus to direct the apoptotic process and the PS display on senescent erythrocytes is the result of the cumulative breakdown of the hemoglobin molecule [1] and is called eryptosis. Normally less than 1% of senescent erythrocytes can be detected in the circulation but in SCD 30–40% of them are senescent [2]. This premature senescence results in many sickle erythrocytes being PS+ and their macrophage elimination being overwhelmed leaving many PS+ erythrocytes in the circulation to eventually be removed by inflammatory necrosis. Before their eventual removal these PS+ erythrocytes adhere to the endothelium via their exposed PS molecules

http://dx.doi.org/10.1016/j.mehy.2015.01.037 0306-9877/Ó 2015 The Author. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

J.R. Kennedy / Medical Hypotheses 84 (2015) 434–436

[3] that also act as platforms for accentuation of blood coagulation [4–6]. Annexin V The physiologic role of annexin V is incompletely understood but what is certain is that it has a very strong affinity for PS and that it has anticoagulant properties. Its PS affinity is such that it’s labeled molecules are used in both the lab and clinically for PS identification. It is a small molecule that is abundant in cells with small amounts present in plasma where it has a brief half-life of +/ 4 min due to its rapid excretion by the kidneys [7]. Its levels are elevated in procoagulant SCD and they increase further during a crisis [8]. A therapeutic role for it in a sickle cell crisis is suggested by an in vitro study showing that it can block the endothelial adhesion of PS+ sickle erythrocytes [3] and its anticoagulant role is dependent on its ability to block the adhesion of blood factors V and VIII to PS displayed on endothelial surfaces [4–6]. The fact that annexin V’s levels are elevated and directly proportional to the severity of coagulation [9] suggests that its primary function is anticoagulation and its anti-adhesive effect in SCD is coincidental. Both the anti-adhesive and anticoagulant properties of annexin V appear to be related to its strong PS affinity and these two properties will be addressed separately. Adhesion in SCD As previously noted 30–40% of sickle erythrocytes are prematurely senescent [2] and unless they are rapidly removed these senescent erythrocytes adhere to the endothelium via a variety of receptors. The significance of this is supported by an in vitro study showing that 36% of the endothelial adhesion of sickle erythrocytes can be prevented by using annexin V to cloak the PS molecules [3] and prevent access to its endothelial receptor thrombospondin (TBS) [10]. The relevance of PS’s adhesive interface with TBS on vascular endothelium is increased by the fact that TBS is also present in plasma where as a multi-domain molecule with many binding sites it can bridge PS+ erythrocytes together and to different molecules on other cells that also bind to TBS. An example of this is the adhesion of PS+ erythrocytes to platelets [11,12] where TBS has binding sites for both the platelet’s CD36 and the erythrocyte’s PS molecules. There is no question that annexin V can block the endothelial adhesion of PS+ sickle erythrocytes [3] but blocking TBS bridging between PS+ erythrocytes and other cells has not been tested. The naturally elevated levels of annexin V that are present in SCD [8] are obviously not preventing sickle erythrocyte adhesion or blood coagulation but may well be modulating both and this will be addressed further during the discussion of coagulation. The coagulation cascade and annexin V During a sickle cell crisis the anti-adhesive ability of annexin V seems likely to be more important in terminating it than its anticoagulant role but you do not have one without the other and attenuation of coagulation pathology during a crisis would be a plus so let’s look at the role of PS and annexin V in coagulation. To examine this role we will first take a very cursory look at the initiation of the propagation phase of blood coagulation. For a clot to form thrombin must be present and damage to the vascular endothelium supplies it via the tissue factor pathway. This thrombin is sufficient to produce a clot and to initiate clot propagation by activating the clotting factor cascade where PS displayed on the surfaces of platelets, erythrocytes, microparticles, monocytes, and endothelial cells serve as platforms for activation of the cascade [4–6] and also act

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as checkpoints for its modulation. Once activated the cascade exponentially increases coagulation by producing more thrombin, a feedback cycle that is unsustainable and dangerous and requires controls two of which are protein C and annexin V. They modulate the cascade at the critical point where thrombin activated factors V and VIII must adhere to endothelial displayed PS in the prothrombinase and tenase complexes in order for the cascade to continue [13–15]. Protein C modulates the cascade by inactivating factors V and VIII [16] while annexin V does so by competing with the factors for access to their endothelial displayed PS. The following in vivo studies support this concept. Coagulation studies in rabbits comparing annexin V and heparin show that annexin V rapidly and safely inhibits fibrin production and platelet aggregation. The first study [17] traumatized carotid arteries and then continuously administered a sufficient amount of these two agents to maintain the blood flow for 180 min. It was found that at the end of that time the annexin V clot weighed 1/3 less, had 47% less fibrin and 42% fewer platelets than the control. In another part of the study when annexin V was mixed with PS+ liposomes before its administration there was no anticoagulant effect. The amount of heparin that was required to maintain blood flow caused significant bleeding but 3 times that amount of annexin V did not. The other study traumatized jugular veins and then gave a single injection of both agents plus radio labeled fibrinogen and found that 4 h later annexin V had reduced fibrin in the clot by 60% while heparin did nothing [18]. This surprising finding is probably the result of both anticoagulants having short half-lives with heparin being rapidly eliminated but with annexin V rapidly attaching to PS+ platelets, microparticles and erythrocytes in the evolving clot. The first study’s demonstration of systemic bleeding with heparin but none with large amounts of annexin V indirectly supports the concept that annexin V’s anticoagulant effect is limited to the coagulation cascade without suppressing the tissue factor pathway. The second study demonstrated annexin V’s ability to rapidly attenuate fibrin accretion by adhering to PS+ platelets and microparticles in the forming clot during its brief time in the circulation.

Conclusion The premature senescence of sickle erythrocytes results in many PS+ erythrocytes being present in the circulation where the exposed PS molecule is responsible for a significant portion of SCD’s adhesive and procoagulant pathology. Approximately one third of sickle erythrocytes display the PS molecule on their surface causing them to adhere to the endothelium and those PS+ erythrocytes and activated platelets in SCD also act as platforms for activation of the coagulation cascade. Annexin V has a strong affinity for PS and by cloaking it both of these pathological conditions can be moderated. In vitro studies show that 36% of the adhesion of sickle erythrocytes is prevented by annexin V [9]. Other in vitro studies show that annexin V prevents the binding of factors V and VIII to endothelial displayed PS in the prothrombinase and tenase complexes [4–6,15] where this binding has to take place before the coagulation cascade is initiated. Annexin V’s modulation of the erythrocyte adhesion and the clotting cascade may well be continuous in SCD because its levels are always elevated there and increase further during a crisis but these beneficial effects appear to always be playing catch up. Perhaps annexin V could use a little help and the infrequent occurrence and transitory nature of a sickle cell crisis suggests that the administration of supra physiologic amounts of annexin V during a crisis may be enough to provide it. Fifty years ago the treatment of a sickle crisis was support and comfort measures and at present it is sophisticated and

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technologically advanced support and comfort measures with nothing to hasten its termination. Annexin V is not the only molecule that blocks a portion of the sickle erythrocyte’s adhesion but it probably is the only one that also modulates its procoagulant state. The safety of its clinical use is suggested by the necessity for PS membrane display in order for it to have any anticoagulant effect, its rapid elimination unless bound to membrane displayed PS (X) and by its normal presence in plasma [8]. Administration of annexin V should have no effect on healthy individuals because of their minimal PS display and if this is found to be the case then clinical trials are in order. Conflict of interest I am the sole author and I have no affiliation with any company in the health care field, have received no income or research support and have no patents regarding this article. References [1] Lutz HU, Bogdanova A. Mechanisms tagging senescent red blood cells for clearance in healthy humans. Front Physiol 2013;4:387. http://dx.doi.org/ 10.3389/fphys.2013.00387 [PMID: 24399969]. [2] Galili U, Clark MR, Shohet SB. Excessive binding of natural anti-alphagalactosyl immunoglobin G to sickle erythrocytes may contribute to extravascular cell destruction. J Clin Invest 1986;77(1):27–333. [3] Setty BN, Kulkarni S, Stuart MJ. Role of erythrocyte phosphatidylserine in sickle red cell–endothelial adhesion. Blood 2002;99(5):1564–71. [4] Thiagarajan P, Tait JF. Binding of annexin V/placental anticoagulant protein I to platelets. Evidence for phosphatidylserine exposure in the procoagulant response of activated platelets. J Biol Chem 1990;265(29):17420–3. [5] Thiagarajan P, Tait JF. Collagen-induced exposure of anionic phospholipid in platelets and platelet-derived microparticles. J Biol Chem 1991;266(36): 24302–7. [6] Ahmad SS, Scandura JM, Walsh PN. Structural and functional characterization of platelet receptor-mediated factor VIII binding. J Biol Chem 2000;275(17): 13071–81.

[7] Ungethüm L, Chatrou M, Kusters D, Schurgers L, Reutelingsperger CP. Molecular imaging of cell death in tumors. Increasing annexin A5 size reduces contribution of phosphatidylserine-targeting function to tumor uptake. PLoS One 2014;9(5):e96749. http://dx.doi.org/10.1371/journal.pone. 0096749. [8] van Tits LJ, van Heerde WL, Landburg PP, Boderie MJ, Muskiet FA, Jacobs N, et al. Plasma annexin A5 and microparticle phosphatidylserine levels are elevated in sickle cell disease and increase further during painful crisis. Biochem Biophys Res Commun 2009;390(1):161–4. http://dx.doi.org/10.1016/ j.bbrc.2009.09.102 [Epub 2009 Sep 30]. [9] Matsuda R, Kaneko N, Kikuchi M, Chiwaki F, Toda M, Ieiri T, et al. Clinical significance of measurement of plasma annexin V concentration of patients in the emergency room. Resuscitation 2003;57(2):171–7. [10] Manodori AB, Barabino GA, Lubin BH, Kuypers FA. Adherence of phosphatidylserine-exposing erythrocytes to endothelial matrix thrombospondin. Blood 2000;95(4):1293–300. [11] Walker B, Towhid ST, Schmid E, Hoffmann SM, Abed M, Münzer P, et al. Dynamic adhesion of eryptotic erythrocytes to immobilized platelets via platelet phosphatidylserine receptors. Am J Physiol Cell Physiol 2014;306(3):C291–7. http://dx.doi.org/10.1152/ajpcell.00318.2013 [Epub 2013 Nov 27]. [12] Wun T, Paglieroni T, Field CL, Welborn J, Cheung A, Walker NJ. Platelet– erythrocyte adhesion in sickle cell disease. J Investig Med 1999;47(3):121–7. [13] Spiegel PC, Kaiser SM, Simon JA, Stoddard BL. Disruption of protein-membrane binding and identification of small-molecule inhibitors of coagulation factor VIII. Chem Biol 2004;11(10):1413–22. [14] Fuentes-Prior P, Fujikawa K, Pratt KP. New insights into binding interfaces of coagulation factors V and VIII and their homologues lessons from high resolution crystal structures. Curr Protein Pept Sci 2002;3(3):313–39. [15] Engelke H, Lippok S, Dorn I, Netz RR, Rädler JO. FVIII binding to PS membranes differs in the activated and non-activated form and can be shielded by annexin A5. J Phys Chem B 2011;115(44):12963–70. [16] Koedam JA, Meijers JC, Sixma JJ, Bouma BN. Inactivation of human factor VIII by activated protein C. Cofactor activity of protein S and protective effect of von Willebrand factor. J Clin Invest 1988;82(4):1236–43. [17] Thiagarajan P, Benedict CR. Inhibition of arterial thrombosis by recombinant annexin V in a rabbit carotid artery injury model. Circulation 1997;96(7):2339–47. [18] Van Ryn-McKenna J, Merk H, Müller TH, Buchanan MR, Eisert WG. The effects of heparin and annexin V on fibrin accretion after injury in the jugular veins of rabbits. J Thromb Haemost 1993;69(3):227–30.

Attenuating a sickle cell crisis with annexin V.

A sickle cell crisis is a painful and dangerous condition that defies effective treatment but fortunately it usually terminates spontaneously and pati...
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