Platelets in Hemostasis and Thrombosis: Role of Integrins And

Transfusion and Apheresis Science 28 (2003) 257–264 www.elsevier.com/locate/transci Platelets in hemostasis and thrombosis: role of integrins 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 Blood Services, Rm 2-006, Bond Wing, 30 Bond St. Toronto, Ont., Canada M5B1W8 Abstract Platelet 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–von Willebrand factor (vWF) interaction may initiate this event, which is followed by firm platelet adhesion mediated by members of the integrin family, such as b1(a2b1, a5b1) and b3(aIIbb3) integrins. In platelet aggregation, although GP1b–vWF, P selectin-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 fibrinogen, 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 thrombin, which then digests and converts Platelets 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 coagulation 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 platelet plug formation is important and subsequent aggregation at the site of vascular in normal cessation of bleeding 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 adhesion 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 collagen, 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]. Fibrinogen 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 thrombus formation [26,27]. veins, the shear rate is low (20–200 sÀ1), but high Understanding these mechanisms has lead to the shear rates are found in arteries (300–800 sÀ1), the use of novel anti-aggregating agents (e.g., the anti- microcirculation (500–1600 sÀ1), or stenotic ves- aIIbb3 antibody ReoPro and the aIIbb3 binding sels (800-10,000 sÀ1) [8]. It is known that the venom peptide Integrilin) that block the GPIIbIIIa platelet membrane glycoprotein (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, von Willebrand disease, 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-glycine-aspartic acid) sequence in ligands (e.g., fibrinogen, vWF, fibro- Integrins are a large family of cell surface re- nectin, vitronectin, 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 protein 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 homology in amino acid 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

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