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Transfusion and Apheresis Science 28 (2003) 257–264 www.elsevier.com/locate/transci

Platelets in and : role of and their ligands Heyu Ni *, John Freedman Transfusion Medicine Research, Department of Laboratory Medicine and Pathobiology, St. Michael’s Hospital, University of Toronto and the Canadian Services, Rm 2-006, Bond Wing, 30 Bond St. Toronto, Ont., Canada M5B1W8

Abstract adhesion and aggregation at the site of vascular injury are two key events in hemostasis and thrombosis. The contribution of several platelet receptors and their ligands has been highlighted in these processes. In platelet adhesion, particularly at high shear stress, GP1b– (vWF) interaction may initiate this event, which is followed by firm platelet adhesion mediated by members of the family, such as b1(a2b1, a5b1) and b3(aIIbb3) integrins. In platelet aggregation, although GP1b–vWF, P -sulfatides, and other molecules, may play some roles, the process is mainly mediated by b3(aIIbb3) integrin and its ligands, such as fibrinogen and vWF. Recent studies with perfusion chambers and intravital microscopy have revised the dogma established with the static (low shear stress) conditions. It is intriguing that platelet adhesion and aggregation do still occur in mice lacking both vWF and fibri- nogen, suggesting that other unexpected molecule(s) may also be important in hemostasis and thrombosis. Crown Copyright Ó 2003 Published by Elsevier Science Ltd. All rights reserved.

1. Introduction providing a membrane surface to enhance gener- ation of , which then digests and converts play a critical role in hemostasis and fibrinogen to fibrin. On the other hand, thrombin thrombosis. They may also contribute to several generated via the process is a potent other physiologic and pathologic processes, such agonist for platelet activation, important for as inflammation, anti-microbial host defense, and platelet adhesion and aggregation [2]. Platelet ac- tumor growth and metastasis [1]. Platelet adhesion tivation and formation is important and subsequent aggregation at the site of vascular in normal cessation of and decrease in injury are one of two key mechanisms required to platelet count [3] or deficiencies of platelet adhe- stop bleeding, the other being mediated by the sion and aggregation are associated with bleeding coagulation system, which generates polymerized disorders [4,5]. In contrast, platelet adhesion and fibrin. There are many interactions between these aggregation may result in thrombosis and vascular two mechanisms leading to clotting. For example, obstruction [6,7]. Unstable angina and myocardial platelets accelerate the coagulation process by infarction are typically the result of platelet adhe- sion and aggregation at the site of atherosclerotic lesions in coronary arteries and thrombosis in * Corresponding author. Fax: +1-416-864-3060. coronary or cerebral arteries is one of the major E-mail address: [email protected] (H. Ni). causes of morbidity and mortality worldwide.

1473-0502/03/$ - see front matter Crown Copyright Ó 2003 Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S1473-0502(03)00044-2 258 H. Ni, J. Freedman / Transfusion and Apheresis Science 28 (2003) 257–264

Therefore, the same processes, platelet adhesion , complement C5b-9, ADP, epinephrine, and aggregation, play critical but contrasting roles thromboxane A2 [19,23]. Platelet activation results (i.e., physiological and pathological). in a conformational change in the aIIbb3 (GPII- bIIIa) receptor on the platelet surface that is then able to bind its major ligand, plasma fibrinogen, 2. Current theory of thrombosis: the processes of resulting in outside-in signaling that furthers platelet adhesion and aggregation GPIIbIIIa receptor clustering and platelet cyto- skeleton reorganization [24,25]. bound Over recent years it has become clear that to activated GPIIbIIIa cross-links adjacent acti- there are differences in the mechanisms of platelet vated platelets resulting in platelet aggregation and adhesion at low and high shear conditions. In hemostatic plug or formation [26,27]. veins, the shear rate is low (20–200 s1), but high Understanding these mechanisms has lead to the shear rates are found in arteries (300–800 s1), the use of novel anti-aggregating agents (e.g., the anti- microcirculation (500–1600 s1), or stenotic ves- aIIbb3 ReoPro and the aIIbb3 binding sels (800-10,000 s1) [8]. It is known that the venom peptide Integrilin) that block the GPIIbIIIa platelet membrane (GP) Ib-IX-V receptor [28,29]. Deficiency of aIIbb3 receptor or complex, together with its ligand, von Willebrand fibrinogen, i.e. Glanzmann thrombasthenia and factor (vWF), is involved in initiating platelet afibrinogenemia respectively, can be associated adhesion to the vessel wall at high shear, such as with severe bleeding. the shear force in coronary arteries [5,6,9–11]. Recently, using a monoclonal antibody to Stable adhesion is then mediated by several aIIbb3 which specifically blocks vWF but not fi- platelet integrin receptors and their ligands. These brinogen binding, it has been confirmed that vWF include the interaction of the a2b1 integrin bound to activated GPIIbIIIa cross-links adjacent (GPIaIIa) with collagen and the aIIbb3 (GPII- activated platelets [30], particularly at high shear. bIIIa; CD41/CD61) integrin interaction with There is also evidence that other molecular pair- fibrinogen and vWF [6,10,12]. On the other hand, ings between platelet receptors and their ligands at low shear, such as in veins, the interactions of may be involved in aggregation. These include the platelet integrins aIIbb3 with fibrinogen or vWF–GPIb complex [31], CD36 and its ligand fibrin, and those of the a2b1 integrin with colla- thrombospondin [32], P selectin–GPIb complex gen, may be able to directly initiate platelet ad- [33], and P selectin–sulfatides [34] interaction. Al- hesion [6,10,13]. Deficiencies of platelet adhesion though P selectin can relocate from platelet a receptors, or of their ligands, have been linked to granules to the platelet surface after platelet acti- bleeding diatheses, e.g. Bernard-Soulier syn- vation [35] and P selectin ligand, PSGL-1, has been drome, , afibrinogenemia demonstrated in the platelet [36], there is no clear [5,14,15]. However, our recent studies with vWF/ evidence that this molecular pair can support fibrinogen double deficient (vWF/Fg-/-) mice platelet aggregation. After potent agonist stimu- demonstrated that platelet adhesion still occurred lation (e.g., by thrombin), released a granule pro- at high shear [16], suggesting that other interac- teins such as fibrinogen, vWF, thrombospondin tions, independent of the GPIb–vWF interaction, [37], fibronectin, and possibly multimeric vitro- occur between platelet receptors and ligands on nectin [38], as well as other granular components the vessel wall [17,18], e.g. GPVI-collagen [19,20], [39,40], may also play an important role in platelet GPV-collagen [21], and GPIb–thrombospondin-1 aggregation [41]. In addition, Matrix Metallopro- interaction [22]. teinases may also enhance platelet aggregation Following platelet-vessel wall adhesion, plate- through outside-in signalling [42]. It is clear today lets aggregate on the layer of adherent platelets to that multiple platelet adhesion molecules are in- form a hemostatic plug. This requires platelet ac- volved in platelet adhesion and aggregation. tivation, stimulated by GPIb–vWF interaction Among them, the integrin families and their and/or various platelet agonists, e.g. thrombin, ligands play pivotal roles. H. Ni, J. Freedman / Transfusion and Apheresis Science 28 (2003) 257–264 259

3. Integrin receptors and their ligands The RGD (arginine--aspartic acid) se- quence in ligands (e.g., fibrinogen, vWF, fibro- Integrins are a large family of cell surface re- nectin, , prothrombin, etc.) is the ceptors. At least 24 integrins have been found in recognition motif of the integrin family [60], and different cell surfaces [43,44]. On platelets, b3 after ligand binding, some divalent cations may (aIIbb3, aVb3) and b1(a2b1, a5b1, a6b1, a8b1) be replaced by an RGD portion of the ligands integrins have been found. Their expression ranges [58,61], but divalent cations are required to from 2–4000 copies (a2b1, a5b1, aVb3) to 50– maintain the specific structure for ligand binding 120,000 copies (aIIbb3) per cell [45]. aIIbb3 is the in most cases [62]. It is important to recognize predominant on the platelet surface, ac- that a key acidic residue (D, aspartic acid) in the counting for about 17% of the total platelet RGD motif is essential for integrin recognition membrane protein [46]. [44]. Some ligand binding sites, such as All integrins contain an a subunit and a b HHLGGAKQAGDV (H12) in the carboxyl ter- subunit and there is in minus of fibrinogen c chain, KGD in Integrilin sequences and similarities of their structures [47]. (snake venom aIIbb3 antagonist) and in human Each subunit polypeptide contains a large extra- CD40L, and QIDSPL in VCAM-1, do not con- cellular domain, a single transmembrane domain, tain the RGD sequence, but they do have a D in and an intracellular domain. A ligand-binding their binding sites [44]. pocket is formed by the extracellular domains of Although the crystal structure of the extracel- both subunits [47]. Bi-directional signals (inside- lular portion of integrin (aVb3) and its RGD out and outside-in) can be transmitted by the two binding form has been recently reported and other subunits, which control integrin conformation integrins may have similar structure [63,64], it is and cell function [43,48,49]. For example, after still not fully clear what biophysical events are ADP stimulation, the signal from ADP receptors happening at the interface between ligands and [50,51] is transmitted to the cytoplasmic tail (i.e., integrins. In the case of fibrinogen-aIIbb3 recog- intracellular domain) of aIIbb3 and in turn nition, three fibrinogen binding sites in aIIbb3 transmitted to the extracellular domain (inside- have been localized, i.e. binding of the C-terminus out), driving a conformational change in the ex- of the chain of fibrinogen to the aIIb b propeller tracellular domain which is then ready to bind its domain (residues 294–314), RGD sequence to b3 ligand. After ligand binding, a signal is trans- (residues 109–180), and the third site to b3 (resi- mitted to the cell (outside-in), which controls cell dues 211–222, the conservative hydrophobic region functions such as morphology change, differenti- in the integrin family) [65]. The last two sites are ation and proliferation, or [25,47]. Both located in the upper face of the I-like domain. It is subunits contain multiple disulfide bonds and not as yet clear which binding site initiates fibri- conformation and ligand binding of aIIbb3 inte- nogen-aIIbb3 recognition in an activated platelet, grin may also be controlled through their disul- but binding of the C-terminus of the chain to the fide bond exchanges [52–54], supporting earlier aIIb may be able to support inactive platelet ad- evidence that dithiothreitol can induce platelet hesion to immobilized fibrinogen [65], suggesting aggregation [55]. Both subunits also contain di- that this interaction may be easier than RGD-I-like valent cation binding sites [56,57]. Three Ca2þ domain interaction. In the in vitro environment, and one Mg2þ have been proposed in the b which may affect structures of both the I-like do- propeller domain of the a subunit; these divalent main and the b propeller domain, the C-terminus cations support one ligand binding site on this of the c chain-aIIb interaction may then become subunit [56]. One divalent cation binding site has absolutely required and fibrinogen becomes the been proposed in the I-like domain (vWF A only ligand for platelet aggregation [66,67]. How- domain-like domain) of the b subunit [57]; this ever, other ligand(s), from either plasma or platelet domain contains an RGD peptide binding site in granules may still be able to support aggregation the up-face of a Rossman fold structure [57–59]. in more physiological conditions, as seen in our 260 H. Ni, J. Freedman / Transfusion and Apheresis Science 28 (2003) 257–264 recent study using an intravital microscopy in fi- and aVb3 may be much less potent in brinogen and vWF/fibrinogen double deficient platelet adhesion and aggregation. mice [16]. b1 integrins (a2b1, a5b1, a6b1, a8b1) are an- Ligands of aIIbb3 (GPIIbIIIa) include fibrino- other family integrins on the platelet surface. Li- gen, vWF, fibronectin, vitronectin, thrombospon- gands of a2b1 include I–IV, , din, collagen, and PECAM-1 [44]. Recently,-Ig6 vitronectin, tenascin [44], and the newly identified [68], prothrombin [69,70], Cyr61 and Fisp12 [71], decorin [80]. Ligands of a5b1 include fibronectin, serum amyloid A [72] and CD40L [73], have been denatured collagen, and L1 adhesion molecule. shown to be possible ligands of aIIbb3; these Ligands of a6b1 include laminin and epiligrin and newly identified proteins may be able to mediate ligands of a8b1 include fibronectin, tenascin and platelet adhesion to an immobilized surface, but vitronectin. These b1 integrin receptors may play there is no evidence indicating that they can sup- an important role in platelet adhesion to injured port platelet aggregation. For aggregation, at least vessel wall, but their roles in platelet aggregation two integrin binding sites are required to cross-link are minor, as they are not able to independently two adjacent platelets and the ligand molecules support platelet aggregation in b3 integrin defi- must be in either the plasma or in the platelet cient platelets. where aggregation occurs. The concentration should be sufficiently high such as to support the low affinity interaction between integrins and their 4. Future directions ligands, e.g. fibrinogen 3 mg/ml, vWF 5–10 lg/ml. appears to be a good candidate to fit The concept of platelet adhesion and aggrega- these criteria (dimer contains two RGD motifs; tion at high shear has been significantly revised by 300 lg/ml in plasma), but there is considerable recent studies using perfusion chambers [10,11,81] evidence that this molecule plays no, or even a and intravital microscopy with targeted ani- negative, role in platelet aggregation [45,74]. mals [16]. Although it is not clear whether platelet Thrombospondin trimer in plasma contains three adhesion to injured vessel wall can still occur, as RGD motifs [75], but its concentration may be too seen in vWF deficient mice, at high shear in type low to support platelet aggregation [76]. Vitro- III vWD patients, it is clear that, in contrast to nectin concentration in plasma is high (450 lg/ml) current theory established under static (low shear but it contains only a single RGD sequence; it may rate) conditions where fibrinogen is required for in fact play a negative role in platelet aggregation platelet aggregation, platelet aggregation does through its occupancy of aIIbb3 [77]. In addition occur in flow conditions in both fibrinogen defi- to the plasma ligands above, fibrinogen, vWF, cient mice [16] and afibrinogenemic humans [11]. thrombospondin, fibronectin, vitronectin, and It remains to be determined which molecules other proteins, may also be secreted from platelet mediate platelet-vessel adhesion at high shear in a granules. These secreted proteins may also play mice lacking vWF. Although several molecular an important role in platelet aggregation. At this pairs are able to support platelet adhesion, in- time it appears that aIIbb3 is required for platelet cluding the b1 integrin family (a2b1-collagen, aggregation, since no platelet aggregation can be a5b1-fibronectin, a6b1-laminin, a8b1-fibronectin), seen in either aIIb [78] deficient or b3 subunit de- the b3 integrin family (aIIb3-collagen, fibrinogen, ficient mice [79]. fibronectin, thrombospondin, aVb3-fibronectin, In addition to aIIbb3, aVb3 (20–40-fold less fibrinogen, collagen, thrombospondin), GPVI- than aIIbb3) is also expressed on the platelet sur- collagen [19], GP V-collagen [21], and GP1b- face [45]. In addition to the ligands mentioned thrombospondin, most of the above mediate above for aIIbb3 (i.e., vWF, fibrinogen, fibronec- adhesion after platelets are tethered by the GPIb- tin, vitronectin, thrombospondin), L1 adhesion vWF interaction. It is unclear whether a single molecules, collagen, osteopontin, and tenascin can molecular pair is sufficient to support adhesion or also bind to aVb3 [44], but interaction of these whether the synergistic action of multiple pairs is H. Ni, J. Freedman / Transfusion and Apheresis Science 28 (2003) 257–264 261 required to support this vWF-independent platelet adhesion and aggregation, or may modulate the adhesion. Identification of these GPIb–vWF- control of thrombosis and hemostasis in normal independent adhesion molecular pairs and their human subjects, i.e. may competitively inhibit roles in human platelet adhesion and platelet ac- overreaction of fibrinogen or vWF mediated ag- tivation will provide new insights into hemostasis gregation and enhance aggregation when either and thrombosis. fibrinogen or vWF is insufficient. It may be asked Platelet aggregation independent of fibrinogen why, if this new protein does exist, has it not been and vWF/fibrinogen in vivo is intriguing. Cross- linked with a bleeding disorder? Explanations may linking two adjacent platelets for aggregation re- be that genetic studies performed have been too quires platelet surface receptors and their ligands. limited or that the new protein(s) may, like fibro- These receptor–ligand pairs include aIIbb3 nectin or other coagulant factors [82–85], be ab- (GPIIbIIIa) and its ligands fibrinogen and vWF. solutely required for embryo development; thus, Several other pairs, e.g. GPIb-IX-V and its ligand no deficient patient survives for study. Identifying vWF, CD36 and its ligand thrombospondin, as and characterizing these proteins will enhance our well as P selectin and its ligands sulfatide and understanding of these novel mechanisms of PSGL-1, also may be involved in this aggregation platelet adhesion and aggregation, contribute to a or in the stabilization of aggregates. However, our fuller understanding of thrombotic pathways, and recent studies with the highly sensitive intravital may subsequently allow for development of new microscope demonstrated that no platelet aggre- antithrombotic and anti-bleeding therapies. gation occurs in mice lacking b3 integrins [16]. This excludes the possibility of other platelet re- ceptors independently mediating platelet aggrega- Acknowledgements tion in vivo, i.e. they are not sufficient in themselves to mediate aggregation, and the b3 H Ni would like to thank Dr Denisa D. Wagner integrin (aIIbb3oraVb3) appears to be the es- (Center for Blood Research, Harvard Medical sential receptor for this process. Interestingly, al- School) for her considerable support and men- though a requirement of fibrinogen for platelet torship during his introduction into the world of aggregation has been documented, and platelet platelet biology. aggregation cannot be induced in vitro if fibrino- gen, or more precisely the QAGDV sequence in the carboxyl terminus of the c chain, is not present References [66,67], we found, using in vivo intravital micros- copy, that very efficient platelet aggregation can be [1] Michelson A. Preface. In: Michelson A, editor. Platelets. seen in mice lacking fibrinogen [16]. This is con- San Diego: Academic Press; 2002. p. xxvii. sistent with recent data from a perfusion chamber [2] Bouchard BA, Butenas S, Mann KG, Tracy PB. Interac- tions between platelets and the coagulation system. In: study of blood from afibrinogenemic patients [11]. Michelson A, editor. Platelets. 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