Platelet Activation by Extracellular Matrix Proteins in Haemostasis and Thrombosis

1358 Current Pharmaceutical Design, 2009, 15, 1358-1372 Platelet Activation by Extracellular Matrix Proteins in Haemostasis and Thrombosis

Steve P. Watson*

Centre for Cardiovascular Sciences, Institute of Biomedical Research, College of Medical and Dental Sciences, Univer- sity of Birmingham, Birmingham, B15 2TT, UK

Abstract: The prevention of excessive blood loss to avoid fatal haemorrhage is a pivotal process for all organisms pos- sessing a circulatory system. Increased circulating blood volume and pressure, as required in larger animals, make this process all the more important and challenging. It is essential to have a powerful and rapid system to detect damage and generate an effective seal, and which is also exquisitely regulated to prevent unwanted, excessive or systemic activation so as to avoid blockage of vessels. Thus, a highly specialised and efficient haemostatic system has evolved that consists of cellular (platelets) and protein (coagulation factors) components. Importantly, this is able to support haemostasis in both the low shear environment of the venous system and the high shear environment of the arterial system. Endothelial cells, lining the entire circulation system, play a crucial role in the delicate balance between activation and inhibition of the haemostatic system. An intact and healthy endothelium supports blood flow by preventing attachment of cells and proteins which is required for initiation of coagulation and platelet activation. Endothelial cells produce and release the two powerful soluble inhibitors of platelet activation, nitric oxide and prostacyclin, and express high levels of CD39 which rapidly metabolises the major platelet feedback agonist, ADP. This antithrombotic environment however can rapidly change following activation or removal of endothelial cells through injury or rupture of atherosclerotic plaques. Loss of endothelial cells exposes the subendothelial extracellular matrix which creates strong signals for activation of the haemostatic system including powerful platelet adhesion and activation. Quantitative and qualitative changes in the composition of the subendothelial extracellular matrix influence these prothrombotic characteristics with life threatening thrombotic and bleeding complications, as illustrated by formation of atherosclerotic plaques or the disorder Ehler-Danlos syndrome, which is caused by a defect in collagen synthesis and is associated with fragile blood vessels. This review will focus on the role of the subendothelial matrix in haemostasis and thrombosis, highlighting its potential as a target for novel anti- thrombotics.

THE COMPOSITION OF THE EXTRACELLULAR vascular matrix are type I and III fibrillar collagens, as ex- MATRIX emplified in the human aorta where they make up 50% of its dry weight [4,5]. These two fibrillar collagens capture circu- The intima extracellular space is localized between the lating VWF and mediate powerful adhesion and activation of endothelial basement membrane and the internal elastic lam- platelets through GPVI and integrin 21 as discussed be- ina representing the border to tunica media. The vascular low [6,7]. Other important components of the vascular ex- endothelial basement membrane (50-100 nm) provides the tracellular matrix include proteoglycans, hyaluronate, elastin primary physical support for endothelial cells and is the first and vitronectin. Elastins are highly abundant in the vascular matrix constituent to be exposed to the flowing blood fol- extracellular matrix, often making up to as much as 50% of lowing denudation of the endothelium. The basement mem- its dry weight, and are essential to preserve elasticity [8]. In brane is primarily composed of laminin (typically 4 laminin addition the proteoglycan chondroitin sulphate contributes to subtypes), which is the most abundant protein, fibronectin, the tensile strength of the vessel wall, particularly in the enactin, several proteoglycans (including heparin sulphate aorta. proteoglycan) and collagen type IV [1-3]. Of these, laminin and fibronectin have been shown to support adhesion and The major components within the vascular extracellular activation of platelets (see below). In contrast, the role matrix that interact directly with platelets are laminin, fi- played by collagen type IV in regulating platelet activation is bronectin, vitronectin and collagen. Thus, the molecular ba- unclear (see below). sis of the interaction of these proteins with platelets is discussed in more detail below. Several other structural proteins The extracellular matrix that lies between the basement also have significant roles in thrombus formation and are membrane and the internal elastic lamina is primarily layed recruited to the extracellular matrix upon damage to the en- down by smooth muscle cells and has been termed the vas- dothelium, including VWF [9], fibrinogen, thrombospondin cular extracellular matrix. The major components of the and proteins involved in the coagulation response, and these

are discussed as well. For a representative structure of an *Address correspondene to this author at the Centre for Cardiovascular arterial wall showing the location of extracellular matrix Sciences, Institute for Biomedical Research, College of Medical and Dental proteins, see Fig. (1) in the Chapter by Eble and Niland (this Sciences, University of Birmingham, Birmingham, B15 2TT, UK; Tel: 00 volume). 44 121 4146514; E-mail: [email protected]

Fig. (1). Steps of platelet adhesion, activation and thrombus formation. At sites of vessel wall damage where ECM is exposed, initial tethering of platelets occurs via GPIb-VWF interaction. VWF is bound to collagen via its A3 domain and interacts with GPIb on the surface of platelets via its A1 domain. Rolling platelets undergo stable adhesion through 21-collagen and IIb3-VWF interaction. At this stage both integrins become activated mainly through inside-out signaling trig- gered by GPVI engagement to collagen. Platelet activation and integrin engagement cause platelet spreading as well as secretion of prothrombotic agents (ADP, TxA2, Fibrinogen). This process recruits additional platelets and stabilizes the growing thrombus. Highly activated platelets within this thrombus provide a ‘procoagulant’ surface leading to massive thrombin generation, which further reinforces platelet activation but also generates a rigid fibrin mesh and leads to clot retraction, which is based on IIb3-fibrin interaction.

THE FUNCTION OF THE SUBENDOTHELIAL MA- The subendothelial matrix is also involved in the regula- TRIX IN HAEMOSTASIS AND THROMBOSIS tion of endothelial cells [12] through integrin-mediated ‘outside-in’ signalling in response to components of the vessel The subendothelial extracellular matrix provides struc- wall such as collagen, laminin, fibronectin, vitronectin and tural support to endothelial cells and is therefore important in fibrinogen. Thus, changes in the composition of the subendo- the organization and maintenance of the shape of the vessel. thelial matrix have the potential to modulate composition The cylindrical shape of the vessel is required for unob- and the response of endothelial cells to other stimuli, includ- structed laminar blood flow. The structural function of the ing shear [13,14]. Activation of endothelial cells induces the extracellular matrix is illustrated by the massive remodeling expression of the pro-thrombotic proteins, VWF, P-selectin that occurs in processes such as restenosis or atherosclerotic and tissue factor, and adhesion receptors, such as E-selectin, plaque development which cause a narrowing of the vessel ICAM-1 and VCAM-1. Expression of VWF and P-selectin lumen [10,11]. This may cause a disturbance to laminar flow on the endothelial surface contribute to the development of and a significant increase in shear force, and eventually lead atherosclerotic plaques through recruitment of platelets and to obstruction of blood flow giving rise to life-threatening leukocytes [15]. Furthermore, once exposed to the flowing thrombotic complications. Indeed, it is noteworthy that blood, the subendothelial matrix plays a critical role in plate- atheroma are frequently found at sites of vessel bifurcation, let adhesion and activation, and thrombus formation, as dis- where flow is disturbed. cussed in further detail below. 1360 Current Pharmaceutical Design, 2009, Vol. 15, No. 12 Steve P. Watson

THE REGULATION OF THROMBUS FORMATION binding to fibronectin and laminin, respectively, although BY THE SUBENDOTHELIAL EXTRACELLULAR this role is redundant with that of integrin IIb3 [23-25]. MATRIX Stable adhesion of matrix proteins to their respective The events underlying thrombus formation at the high integrins serves to increase the net affinity of collagen for shear rates of the arterial system that occur following vascu- GPVI leading to enhancement of signals through the immu- lar damage or rupture of atherosclerotic lesions have been noglobulin receptor. In addition, clustering of the integrins subject to intensive research over a number of years, and themselves generates outside-in signals that serve to rein- have led to the development of a multistep model. The trig- force platelet activation, albeit these are much weaker than ger is the denudation of endothelial cells causing exposure of those of GPVI [21]. Thus, platelet glycoprotein receptors the subendothelial extracellular matrix to the flowing blood. generate a repertoire of signals that mediate platelet adhesion This is followed by capture (or tethering) of platelets, which and spreading at sites of vessel injury, with the latter serving is a specialized process that serves to counter the very high to strengthen platelet adhesion. shear rates of the arterial system, including those found at The secretion of platelet dense granules and activation of sites of stenosis. Tethering of platelets is accomplished by phospholipase A leads to the release of ADP and formation binding of VWF to subendothelial collagen which leads to a 2 of TxA2, respectively, the two major feedback mediators of conformational change that enables it to bind to the GPIb- platelets. These two agonists synergise with each other and IX-V complex on the platelet surface (for review [16]). The with signals from platelet glycoprotein receptors to mediate interaction between VWF and GPIb-IX-V has a very fast on- further platelet activation. Crucially, the two feedback mes- rate of association that leads to the formation of an effective sengers, along with VWF and fibrinogen, mediate formation bond at high shear and therefore platelet capture [17]. Strik- of a platelet aggregate through a continual cycle of platelet ingly, the effectiveness of the VWF conformational change tethering (via VWF), platelet activation (primarily via ADP and subsequent binding to GPIb binding increases with in- and TxA2) and platelet crosslinking (via fibrinogen or VWF creasing shear. The interaction between VWF and GPIb-IX- binding to integrin IIb3). Moreover, this scheme of events V has a very fast off-rate, however, thereby leading only to is further enhanced by generation of thrombin through the the transient capture of platelets [17]. This is illustrated by coagulation cascade which activates platelets through bind- the rolling of platelets in the direction of flow on a mono- ing to the protease activated receptors, PAR1 and PAR4, as layer of VWF, with stable aggregates only being formed well as converting fibrinogen to fibrin, which serves to fur- after several minutes through the weak activation of integrins ther strengthen the growing thrombus. The growing throm- by the GPIb-IX-V complex [18]. bus is also consolidated by low affinity interactions between The tethering of platelets to VWF enables other matrix several platelets surface proteins, including binding of CD40 proteins with a slower on-rate of association to their recep- ligand (CD40L) to integrin IIb3, and binding of ephrins to tors to interact with platelet surface receptors. Crucially, Eph kinases. Furthermore, thrombus formation is completed these interactions would not occur in the absence of VWF by an integrin-driven clot retraction, a process through which [19]. The most important interaction is that of the fibrillar contraction of the actin-myosin cytoskeleton is channelled collagens, type I and III with the low affinity immunoglobu- via integrins to fibrin [26]. lin surface receptor, GPVI. Clustering of GPVI by collagen Alongside the above events, platelets provide a ‘proco- or by laminin, which has an approximate tenfold lower affin- agulant’ surface through exposure of phosphatidylserine ity for the glycoprotein receptor compared with collagen leading to a burst of thrombin generation, which further rein- [20], generates powerful signals that lead to activation of forces platelet activation and generates fibrin, leading to clot platelets, including inside-out activation of platelet integrins, retraction, as described above. A simultaneously activated cytoskeletal remodeling (leading to platelet spreading), gran- fibrinolytic response prevents excessive thrombus formation ule secretion and thromboxane A2 (TxA2) formation [6]. Ac- (for review [27]). tivation of integrins converts the tethering of platelets to stable adhesion. In contrast, the affinity of the interaction be- The above model emphasizes that platelet adhesion to the tween collagen or laminin with GPVI is not sufficient to me- subendothelium represents the first step in thrombus forma- diate stable adhesion. The platelet integrins that interact with tion following damage to the arterial system. The early the extracellular matrix are IIb3, 21, 51, 61 and events in this cascade have attracted much interest for phar- v3. Of these, the most important two are considered to be macological intervention in anticipation that this may over- IIb3, which is expressed at 80,000 - 120,000 copies per come limitations of current ant-platelet regimes. In this re- platelet, and 21, which has 1500 - 3000 copies. Integrin view, we shall discuss what is known about the structural IIb3 mediates stable adhesion through binding to VWF features of the major matrix proteins that mediate platelet and fibronectin, and also mediates platelets aggregation binding and platelet activation, as well as the possibility of through interaction with above ligands and also fibrinogen pharmacological intervention and therapeutic potential. [21]. Integrin 21 mediates stable adhesion through binding to collagen, although this role can be masked by the in- COMPONENTS OF THE VASCULAR EXTRACEL- teraction of VWF with integrin IIb3. Thus, the importance LULAR MATRIX AND PLATELET ACTIVA- for 21 in supporting adhesion to collagen is increased TION/ADHESION under conditions of reduced VWF plasma concentration A). Collagen [22]. Integrins 51 and 61 have also been shown to support stable adhesion of platelets under high shear through There are at least 43 distinct collagen chains in the human genome, assembling into 28 collagen types [28]. Platelet Activation by Extracellular Matrix Proteins Current Pharmaceutical Design, 2009, Vol. 15, No. 12 1361

Their common feature is the Col-domain, a triple-helical proach based on the use of toolkits of triple-helical peptides core. Three polyproline helices form a superhelical structure, (summarized in [38]). From these very elegant studies, it has characterized by the sequence glycine-X-Y. This sequence become clear that although 21 binds with highest affinity may be repeated more than 300 times, so that a triple-helical to GFOGER (single amino code), it binds with varying af- domain is often more than 1,000 amino acids in length. The finities to more general sequences of GXXGER, thus allow- chains form a right-handed triple helix and may be identi- ing for a controlled adhesion and cell migration over a colla- cal or different, depending on the type of collagen. The X- gen surface. For GPVI it has become clear that the spacing position is often taken by proline (Pro), while the Y position of the GPO-triplets plays a crucial role in GPVI-collagen is frequently occupied by 4-hydroxyproline (Hyp) [29]. This interaction [39]. The VWF binding sequence on collagen has regular repetition and high glycine content is found in very been identified as GPRGQOGVMGFO [40]. The intact se- few other fibrous proteins. Glycine, being the smallest amino quence is found in collagen types II and III, but not in intact acid, is required at every 3rd position where it occupies the collagen type I. However, VWF binding to type I collagen interior of the helix, since there is no space for side chains. has been shown. It has been suggested that a binding region The rings of Pro and Hyp point outward, thermally stabiliz- for VWF on collagen type I could be formed by contribu- ing the triple helix. More than 90% of the collagen in the tions of both (1 and 2) chains [38]. body is type I, II, III and IV. The first step in platelet adhesion and activation follow- Collagen Types in the ECM ing damage to the vessel wall is the interaction of plasma VWF with subendothelial collagen under shear, as discussed While collagen types I, III, IV, V, VI, VIII, XV, XVIII above [41]. Platelets are tethered by the interaction of the and XIX have been found in the vessel wall, types I, III and GPIb-IX-V complex with the A1 domain of VWF, which is V are the most prominent [30]. Collagen I and III form fi- exposed by shear [42,43]. VWF is immobilized on collagen bres, confer tensile strength as well as flexibility. Collagen V types I and III in the subendothelial matrix via its A3 domain is found as co-polymer in association with other collagens [44,45]. In addition, the A1 domain has also been reported to and may also form filaments by itself. Collagens IV and mediate binding to collagen type VI in the subendothelial VIII, forming 2D or 3D networks, are believed to lie closer matrix [46,47]. Platelets roll over the collagen-VWF surface, to the actual vessel lumen than other collagens [4,31]. Colla- forming new GPIb-VWF bonds at the front while releasing gen type IV is found directly beneath the endothelial cells in bonds at the tail. During rolling under high shear conditions, the vascular endothelial basement membrane, often in asso- platelets also shed parts of their membranes, which are in ciation with laminin around smooth muscle cells. Collagen firm contact with VWF leaving trails of procoagulant mi- type IV is a non-fibrillar collagen and is considerably less croparticles [48]. Firm attachment of platelets to collagen is potent than the fibrillar collagens in supporting activation of achieved via the 21-collagen interaction or indirectly via the major signalling receptor for collagen on platelets, gly- binding of VWF to integrin IIb3. Both integrins have to coprotein VI (GPVI), or supporting adhesion through re- be in an active conformation to mediate binding. Activation cruitment of von Willebrand factor (VWF) [32]. On the other is mediated by binding of talin and kindlin-3 to their cytoso- hand, there are reports that collagen type IV is able to sup- lic tails (inside-out signalling) following agonist-induced port GPVI-mediated platelet aggregation in a low shear envi- platelet activation [49,50]. Collagen is the most powerful ronment, or platelet adhesion and thrombus formation at agonist in the subendothelial matrix mediating activation, arteriolar rates of flow [33,34]. The explanation for these although weak signals from GPIb-IX-V also contribute to contradictory observations may be related to the method of activation. collagen preparation. Thus, the importance of basement membrane collagen type IV in supporting platelet activation The collagen-binding region of 21 is the I-domain, and thrombus formation warrants further investigation. Fur- also called A-domain due to its analogy to the vWF A1- ther into the ECM, collagen VI is found, forming beaded domain [51]. Crystallography, using mutant 2 I-domains, filaments. Following superficial vessel wall damage, these has enabled identification of the critical residues that support sub-endothelial collagens are the first ones to contact patrol- binding of a synthetic collagen triple helix peptide contain- ling platelets. On the other hand, increased damage reaches ing GFOGER [52]. The glutamate coordinates the divalent 2+ the internal elastic lamina and deeper layers of the media, cation (under physiological conditions Mg ) in the integrin exposing collagens type I and III and also tissue factor which metal ion dependent adhesion site (MIDAS). The MIDAS is activates the coagulation cascade. not exposed in the inactive conformation and only becomes accessible to collagen l upon integrin activation [53]. Helix 7 Collagen I is present in abundance in the cap of plaques of the integrin undergoes a dramatic movement during acti- [35]. Remodelling of the vessel wall, including collagen vation suggesting that this is a mechanism for affinity regu- turnover, induced by proteolytic activity of macrophages, is lation of the integrin leading to agonist binding and possibly important for the ability of smooth muscle cells to migrate also outside-in signalling, which involves clustering of the and proliferate within the intima. This contributes to the de- integrin [54,55]. Further regulation of integrin 21 is velopment of plaques. achieved via rearrangement of disulphide bonds, catalyzed Direct and Indirect Binding of Platelets to Collagen by protein disulphide isomerases on the platelet surface [56], although this remains controversial. Studies with recombi- The main collagen receptors on platelets are considered nant collagen peptides have shown that only GFOGER to be the integrin 21 (VLA-2, GPIa/IIa) and GPVI [36,37]. (found in collagen type I, II and IV) is capable of binding to Over the last years the binding regions of collagen receptors the inactive conformation of integrin 21 [57]. The binding on different types of collagen have been analyzed by an ap- to the other GXXGER sequences depends upon inside-out 1362 Current Pharmaceutical Design, 2009, Vol. 15, No. 12 Steve P. Watson signalling. The main role of integrin 21 in platelet colla- an increased expression of the Lutheran glycoprotein, sickle gen interaction is to support firm adhesion, with GPVI play- cells have been shown to have increased affinity for binding ing a crucial role in platelet activation. to laminin relative to normal red cells, possibly contributing to vasoocclusive episodes in these patients [75,76]. Other Studies using mice deficient in the GPVI signal transduc- laminin receptors include dystroglycan, a heterodimeric tion subunit, the Fc receptor chain, or treatment of mice polypeptide particularly important for the function and struc- with a GPVI-depleting monoclonal antibody, JAQ1, suggest ture of muscle fibres, the ‘collagen’ receptor GPVI and an that under certain experimental conditions, GPVI is the only uncharacterised platelet protein of 67 kDa receptor [20,77, collagen receptor of importance in thrombosis [58]. Indeed, 78]. treatment with JAQ1 leads to a complete loss of the thromboembolism induced by collagen and epinephrine. On the Laminin - Platelet Interactions other hand platelets from 1-deficient mice develop less stable aggregates under flow, pointing to an important role Adhesion of platelets to laminin was first described in of 21 in thrombosis [59]. Experimental and species (e.g. 1983 and was subsequently shown to be due to binding to mouse platelets are much smaller than human) differences integrin 61 (also known as VLA-6) [79-81]. The in- might be one reason for the sometimes conflicting results. volvement of GPVI in laminin- induced platelet activation However, the focus on certain aspects of artificial thrombus was only recently reported [20]. The 67 kDa laminin recep- formation might lead to overestimation of the role of the two tor, that was first identified on human breast carcinoma and receptors, as exemplified in the study of Auger et al. This muscle cells [82,83] is also expressed on platelets, although study suggested two distinct mechanisms underlying stable almost nothing is known about its function [81,84,85]. Thus, adhesion to collagen, namely via VWF bridging or through it seems that the principle laminin receptor on platelet is the direct binding, possibly via integrin 21 to the GFOGER integrin 61, while GPVI functions as a downstream recep- motif, as this does not require inside-out activation of the tor, contributing to laminin induced platelet activation. In the integrin [60]. absence of GPVI platelets are still able to adhere normally to laminin but spreading and lamellipodia formation are B). Laminin strongly reduced [20]. Biacore studies have revealed that the interaction between laminin and GPVI has a ten-fold lower Laminins are heterotrimeric glycoproteins consisting of affinity compared to binding to collagen. This lower affinity three polypeptide chains (, , ) [61,62]. 5 genes encode for may explain why 61 binding is a prerequisite for GPVI the -chains and 3 each for the - and -chain [62]. The binding and why laminin, in contrast to collagen, is unable to chains can assemble in various combinations and 15 differ- induce significant tyrosine phosphorylation or platelet ag- ent laminin subtypes have been described [2]. The expres- gregation in suspension [20]. The concept of 61 and GPVI sion of the different laminin subtypes is tissue specific. as the platelet laminin receptors is very appealing since it Basement membranes of all blood vessels are characterized mirrors the cooperation of 21 and GPVI in the interaction by the presence of 4 laminin subtypes [61]. of collagen [6]. The central part of the laminin molecule is characterized Again in analogy to platelet-collagen interaction, VWF is by a triple -helical assembly of the , , and chain [63]. required for platelet adhesion to laminin under flow condi- While the C-terminal parts of the and -chain are within tions [24]. Anti-VWF antibody treatment blocks platelet ad- the triple-helical assembly, the -chain extends further to hesion to laminin completely under high (1500 s-1) but not form five globular motifs, called g-domains. This domain is under low shear (300 s-1) conditions. Similar results were an important mediator of laminin - cell interaction because obtained with VWF deficient blood (type III VWF disease) of its involvement in binding to various integrins [62,64]. or blood from Bernard-Soulier syndrome patients (GPIb de- The N-terminus of the molecule is not triple helical and con- ficiency) [24]. Therefore, in whole blood under flow condi- sists of short arms of the 3 chains, forming distinctive do- tions, the interaction of VWF with laminin seems to be the mains. These domains are critical for the self-assembly of initial step. Recruitment of platelets by VWF may enable the laminin into the polymer that is found in endothelial integrin 61 and GPVI to interact with laminin and thereby basement membranes [65-67]. support integrin activation giving rise to stable adhesion and Laminin Receptors formation of small aggregates. The interaction of VWF with components of the extracellular matrix other than collagen The integrins 11, 21, 61, 64, 71, 91 and has been suggested for several years [86,87], but laminin is v3 have been shown to interact with laminins, with differ- the first non-collagen to be able to mediate direct binding of ent preferences for the various laminin subtypes. However, VWF, a result that has been confirmed by Biacore [24]. the only reported laminin-binding integrin on the platelet In conclusion, laminin seems to interact with platelets surface, 61, is known to bind all laminins subtypes both indirectly via VWF and directly through integrin 61 [68,69]. On the other hand, 21 does not appear to support and GPVI. The role of the 67kDa protein in the interaction of binding of platelets to laminin [70,71]. laminin with platelets is not known. The sequence of events Several additional cell surface molecules on platelets and that results in adhesion of platelet to a laminin surface under other cell types have also been described to interact with flow conditions is similar to that seen on collagen. The over- laminin. Among them is the Lutheran glycoprotein, a blood all significance of the interaction of platelets with laminin group glycoprotein expressed on erythrocytes [72]. The Lu- however, would appear to be minimal due to collagen expo- theran glycoprotein mediates adhesion of red cells to sure. On the other hand, it can be speculated for example that laminin, specifically to 5-chain laminins [73,74]. Based on the interaction of platelets with laminin plays a key role fol- Platelet Activation by Extracellular Matrix Proteins Current Pharmaceutical Design, 2009, Vol. 15, No. 12 1363 lowing superficial injuries that lead only to disruption of the sequence [110], although additional IIb3-binding sites in endothelial lining or during the final stages of angiogenesis fibronectin have been identified. This multisite interaction of as a mechanism for completing the endothelial monolayer IIb3 with fibronectin seems to be necessary for a tight through release of growth factors. In such cases the deeper, interaction [111], with RGD-mediated integrin binding being underlying collagen may not be exposed. essential for platelet adhesion under shear to fibronectin [112]. Platelets also express two additional fibronectin recep- C). Fibronectin tors 51 and v3 [79,113]. 51 has been shown to have non-redundant role in platelet adhesion to fibronectin, since Structure of Fibronectin antibodies to 5 are able to inhibit platelet deposition under Fibronectin is a homodimeric glycoprotein consisting of flow (300s-1) by approximately 50%. In comparison RGD- 250 kDa subunits linked by two disulphide bonds in their containing peptides or antibodies to IIb3 blocked this in- COOH-termini [88]. In contrast to laminin and collagen, teraction by over 85% [25,112]. fibronectin is present in blood plasma at a concentration of While IIb3 supports platelet adhesion as well as acti- 300-400g/ml (~10% of the fibrinogen concentration) as vation in response to fibronectin, 51 and v3 seem to well as in platelet -granules [89-91]. Fibronectin RNA is mainly function as adhesion receptors. Platelets adhere and alternatively spliced at three sites generating EIIIA(EDA), spread on fibronectin coated surfaces under static conditions EIIIB(EDB) and V (CS-1) variants. EIIIA and EIIIB variants [80,114,115]. Platelet adhesion to fibronectin induces forma- are mostly found in the extracellular matrix of blood vessels, tion of filopodia and lamellipodia through binding to integrin but are also present in platelet granules [92,93]. In contrast IIb3, whereas binding to integrin 51and v3 induces plasma fibronectin mostly lacks these domains, although the 2+ only filopodia formation [23,116]. Robust Ca mobilization level of EIIIA fibronectin in plasma seems to be increased in is dependent on the interaction of IIb3 with fibronectin disease states associated with an increased thrombotic risk [23], causing outside-in signaling as observed for the binding such as atherosclerosis, diabetes and stroke [94-96]. Plasma of fibrinogen to integrin IIb3 [117]. Interestingly, adhe- fibronectin is a soluble dimer, while extracellular matrix sion of platelets to fibronectin is not inhibited in the absence fibronectin is assembled into insoluble aperiodic matrix fi- of functional IIb3, supporting a role for 51 and v3 in brils. The latter is a cell-mediated process, under involve- this process [23,113]. However, neither 51 nor v3 are ment of integrins and cell surface LAMMs (molecules of able to induce a similar level of spreading to fibronectin in large apparent molecular mass). Cells dedicated to fi- comparison to IIb3, which may be due to an almost two bronectin assembly are fibroblasts, vascular smooth muscle orders of magnitude higher expression level of the latter. cells and endothelial cells but also platelets [97]. Further- more, several binding sites for adhesive proteins such as fi- Although all these studies support a function of purified brin, collagen, VWF and integrins have been identified in the fibronectin in platelet adhesion and spreading, an important fibronectin molecule, underlining its capability to crosslink question is the relative role of fibronectin within the suben- molecules and cells [97]. dothelial extracellular matrix, particularly taking into ac- count the presence of other platelet-regulatory proteins. This Fibronectin Receptors has been addressed using human umbilical artery subendo- Cell surface receptors for fibronectin belong to the in- thelium and antibodies against fibronectin [118]. Incubation tegrin family and several different integrins have been de- of the subendothelium with an anti-fibronectin antibody re- scribed to bind fibronectin (31, 51, 41, 47, sulted in a significant inhibition of platelet adhesion. Inter- IIb 3 and v 3) [98-103]. While most of the above in- estingly, this inhibition is dependent on the wall shear rates -1 tegrins interact via a characteristic RGD- containing se- used, being most pronounced at shear rates above 800s and -1 quence, 41 and 47 bind to a site characterized by a absent at 400s [118]. In contrast to the situation on collagen LDV motif [104]. Of the above, only 51, IIb3 and or laminin, VWF is not able to bind fibronectin and therefore v3 are expressed on platelets. does not mediate platelet adhesion to fibronectin under flow. The Interaction of Cell Matrix Fibronectin with Platelets Fibronectin regulates platelet activation and thrombus formation at several checkpoints. Soluble, plasma fibronectin The interaction of fibronectin with platelets and a role for significantly contributes to thrombus stability and thrombus subendothelial fibronectin in the regulation of platelet re- growth (see below). Fibronectin within the endothelial ex- sponses has been suggested since the late seventies, although tracellular matrix also supports platelet adhesion and spread- this still remains poorly understood [105,106]. This is in part ing [25]. Interestingly, this interaction seems to be more im- due to the presence of plasma fibronectin and the embryonic portant under high shear conditions [118], typically reflect- lethality of the fibronectin-deficient mice [107], as these ing a pathological situation of a stenosed artery. have hampered investigation of the role of extracellular matrix fibronectin in thrombosis and haemostasis. D). Vitronectin The first evidence for the presence of fibronectin recep- Structure of Vitronectin tors on platelets came from the observation that platelets from a patient with Glanzmann’s thrombasthenia showed a Human vitronectin is a 75kDa protein which has a similar strongly reduced binding to fibronectin [100,108]. Subse- distribution to fibronectin [119]. Thus, it can be found in quently, by using chemical cross-linking and monoclonal plasma (200-500g/ml), in the extracellular matrix and in antibodies, IIb3 was shown to bind fibronectin directly platelet -granules where it is taken up from the plasma [109]. Binding of fibronectin to IIb3 involves the RGD [120-122]. 1364 Current Pharmaceutical Design, 2009, Vol. 15, No. 12 Steve P. Watson

Vitronectin Receptors injury and collagen/epinephrine induced thromboembolism, although the fibronectin plasma levels are only 20% of con- Vitronectin binds many of the proteins involved in trol mice. The enhancement of collagen/epinephrine induced thrombosis and haemostasis, including plasminogen activa- thromboembolism strongly supports the role for plasma EI- tor inhibitor-1 (PAI-1), plasminogen and heparin. Vi- IIA fibronectin, since in this model no vessel wall injury is tronectin posseses a RGD site that enables it to bind to involved. Interestingly, venous thrombosis as well as coagu- IIb 3 and the minor platelet integrin, v 3 [123]. How- lation parameters were unchanged in these mice, strongly ever, the role of integrin v 3 in supporting platelet activa- suggesting that the prothrombotic effect is based on an al- tion is unclear. Moreover, the ability of vitronectin to bind to tered EIIIA fibronectin - platelet interaction [131]. Although integrin IIb 3 is masked by the presence of other ligands, the molecular basis of this enhanced thrombosis is not clari- including fibrinogen, fibronectin and VWF. fied, enhanced EIIIA fibronectin plasma levels may repre- The Interaction of Cell Matrix Vitronectin with Platelets sent a risk factor for thrombotic complications in certain pathologies [94-96]. The interaction of platelet IIb3 and v3 with cell matrix associated vitronectin may well have a supportive Thus, it seems that fibronectin present in the plasma function in platelet adhesion to the subendothelium. How- and/or platelet granules contributes to thrombus stabilization ever, experimental data to support this hypothesis are, to our in the presence of VWF and fibrinogen. Possible mecha- knowledge, not available at the moment and studies investi- nisms are the support of platelet - platelet contacts via in- gating the role of vitronectin in thrombosis and haemostasis tegrins [132] as well as stabilizing the fibrin network and are mainly focused on the plasma fraction. cross-linking of platelets to fibrin [133,134]. Another process that may support thrombus formation THE ROLE OF MATRIX PROTEINS PRESENT IN and stabilization is the assembly of plasma fibronectin into PLASMA AND PLATELET -GRANULES fibrillar extracellular matrix arrays, a process observed on VWF and fibrinogen are the two major plasma proteins several cell types including activated platelets [135-137]. supporting platelet adhesion to the extracellular matrix and Thus, activated, adherent platelets are able to assemble platelet thrombus formation through the formation of platelet plasma fibronectin into an insoluble matrix on their surface - platelet contacts. However, unexpectedly mice deficient in which may support platelet cohesion and therefore thrombus both proteins are still able to form occlusive thrombi, al- strength. The importance of this process was shown using though with strongly delayed kinetics [124]. This demon- different proteins specifically interfering with this process. In strates that other plasma proteins or platelet granular compo- flow chamber experiments, inhibition of fibronectin assem- nents are able to substitute for VWF and fibrinogen and may bly resulted in a decrease of thrombus volume and the num- therefore also play a role in thrombus formation. ber of adherent platelets by 50-60% [134]. Plasma fibronectin is clearly an important player in Plasma Fibronectin thrombus formation, although its precise role remains unde- Early experiments investigating platelet thrombus forma- fined. Last, but not least, this is emphasised by the observation under flow indicated a supportive role of plasma fi- tion that increased fibronectin plasma levels are associated bronectin in thrombus formation. Using collagen-coated sur- with an increased risk of coronary artery disease [138,139]. faces, platelet adhesion as well as thrombus formation was reduced in fibronectin-depleted plasma. Interestingly, the Vitronectin diminished thrombus formation in fibronectin-depleted Depending on the in vivo model and/or vascular bed, plasma was present at low and high shear rates [125-127]. In vitronectin-deficiency has a variable effect on thrombus vivo investigations also support a role for fibronectin, as formation. Thus, under certain conditions, there is increased mice deficient in VWF and fibrinogen form occlusive embolization, reduced thrombus growth and stability and thrombi. Fibronectin was proposed as the likely substituting therefore increased time to vessel occlusion in vitronectin- candidate protein because of its structure and its ability to deficient mice [140-142]. Importantly, early platelet adhe- interact with IIb3 [128,129]. Moreover, a role for fi- sion to the injured vessel wall was not altered in these mice bronectin in thrombus formation in vivo was also shown in [140], indicating that extracellular matrix vitronectin is not mice expressing less than < 2% of the plasma fibronectin and involved and that other components of the extracellular ma- < 20% of platelet fibronectin. These animals showed delayed trix are able to compensate. This suggests that the deficiency thrombus formation, increased embolization and an in- of plasma vitronectin is the cause for the phenotype. creased time to occlusion in the presence of normal amounts of fibrinogen and VWF [124]. The importance of fibronectin The major role of plasma vitronectin in regard to haemo- was further substantiated in heterozygous mice, having 50% stasis may be its ability to bind to plasminogen activator in- of the normal fibronectin levels. This reduction was suffi- hibitor (PAI-1). PAI-1 is the major physiological inhibitor of cient to have a profound inhibitory effect on thrombus for- tissue-type plasminogen activator (tPA) and therefore an mation in vivo following arterial injury [130]. In a very re- instrumental regulator of plasmin and fibrinolysis. Vi- cent study Cauhan et al. showed, that the presence of a spe- tronectin binds PAI-1, stabilizes the active confirmation of cific fibronectin splice variant (EIIIA) in plasma may have the inhibitor and thereby increases the activity of PAI-1 strong prothrombotic effects. Mice engineered to express [143-145]. This ultimately leads to a reduced fibrinolytic EIIIA fibronectin only, have strongly accelerated thrombosis response and enhanced thrombus formation. Thus, vi- in two different in vivo thrombosis models, ferric-chloride tronectin deficiency should cause reduced PAI-1 activity and Platelet Activation by Extracellular Matrix Proteins Current Pharmaceutical Design, 2009, Vol. 15, No. 12 1365 enhanced fibrinolysis, which nicely corresponds to the ob- exposure) in the pathway suggest an effective and more served phenotype of the knockout mouse (increased emboli- uniform prevention of thrombus formation. zation, reduced thrombus growth and stability). Interestingly, iii) Thirdly, several mechanisms seem to be more important the thrombotic phenotype of vitronectin and PAI-1 double- under the high shear conditions that are typically found in deficient mice is indistinguishable from vitronectin or PAI-1 stenosed arteries. Thus, selective interference of platelet deficient mice [142]. This strongly suggest that both proteins activation in pathological high shear situations may lead serve the same mechanism, and that other vitronectin func- to only minimal disturbance of normal haemostasis. tions in thrombus formation are not significant, at least under the experimental conditions used. The connection between iv) Early platelet activation and adhesion is instrumental for PAI-1 and vitronectin is further emphasised by their co- the role of platelets in atherosclerosis development. localisation in platelet granules [146,147]. Release of PAI-1 It is important to note, that most of the currently used into the thrombus by activated platelets inhibits fibrinolysis, platelet inhibitors (phosphodiesterase inhibitors, the cy- while simultaneous liberation of vitronectin stabilises this clooxygenease inhibitor aspirin and P2Y12 antagonists) do process based on the mechanism described above. not interfere directly with these early events but are targeting Thus, based on these data, plasma vitronectin is impor- late mechanisms or secondary platelet activation. Although, tant for the regulation of thrombosis, most likely by regula- IIb3 antagonists can affect primary adhesion their main tion of PAI-1. The contribution of vitronectin associated function being the inhibition of the final common pathway of with the extracellular matrix seems to be minor, although platelet aggregation. Thus, several different approaches have this has to be said with caution since few data are available. been investigated to interfere with primary platelet activation and adhesion: PHARMACOLOGICAL INHIBITION OF PLATELET - EXTRACELLULAR MATRIX INTERACTION AS Collagen ANTI-THROMBOTIC MECHANISM The early steps of platelet-collagen interaction are seen The critical role of platelets in arterial thrombosis has as highly promising for anti-thrombotic intervention. None been well established. Furthermore, it becomes more and of the currently marketed anti-thrombotics target these more evident that platelets also play a significant role in events, last not least due to the fact that protein-protein inter- other pathological processes such as atherosclerosis [148]. actions are not easy to inhibit, especially with small mole- Thus, anti-platelet regimes belong to the standard treatment cule compounds. in patients with atherothrombotic disease for acute and pro- Biologics interacting with collagen that show some in- phylactic prevention of thrombotic events. Landmark clinical hibitory effect on platelet binding in model settings include studies for aspirin or clopidogrel have shown the enormous the recombinant leech proteins rLAPP (from Haementeria benefit of anti-platelet agents for the prevention of officinalis) and saratin (from Hirudo medicinalis), the mos- atherothrombotic events [149-151]. Moreover, dual anti- quito salivary gland protein aegyptin (from Aedes aegypti) platelet therapy combining aspirin and the P2Y12 antagonist as well as CTRP-1, a C1qTNF-related protein from plasma clopidogrel has been shown to further reduce the risk of [155-158]. All of them have been shown to act in vivo as atherothrombotic complications, with a 20% relative risk inhibitors of platelet function. The exact collagen binding reduction in patients with unstable angina or non - ST myo- site of LAPP is not known. For saratin there is interference cardial infarction as compared to aspirin mono-therapy seen with both VWF and 2-I-domain binding to collagen, [152]. Despite this significant achievement, a high number of suggesting a certain overlap in binding epitopes on collagen patients experience thrombotic events while under anti- [159]. Recently the 3D structure of saratin was solved. The platelet treatment. Atherothrombosis is still a leading cause data from docking experiments with collagen model peptides of death worldwide and, importantly, the absolute number of lead to the conclusion that despite a similar global fold of deaths from this disease continues to increase [153,154]. LAPP and saratin there is a significant difference in collagen Thus, there is a large unmet clinical need and much effort is binding [160]. CTRP-1 blocks the binding of vWF-A3 do- put into the improvement of existing drugs, e.g. the devel- main to collagen and thus inhibits collagen-induced platelet opment of new P2Y12 inhibitors, but also in the exploration aggregation [157]. Aegyptin has been shown to block the of new platelet (thrombus) - inhibitory mechanisms. In this interaction of the three glycoprotein receptors, GPIb, GPVI regard interference with primary platelet activation and ad- and 21 with collagen, thereby showing that it is distinct hesion to the extracellular matrix is considered to be a poten- from the other molecules described above [156]. Thus it tially attractive approach. This is based on the following as- might serve as a powerful tool to evaluate the relative impor- sumptions and supporting experimental data. tance of all three receptors in the very early steps of platelet i) Inhibition of the initial step of platelet activation prevents adhesion and activation. Targeting collagen itself would formation of activated platelets. This mechanism has the limit the action of an inhibitor to sites where collagen is ex- potential to be very effective in blocking thrombus for- posed but also limit the use of such inhibitors for systemic mation and growth. application. ii) The interaction of GPIb and VWF, and of GPVI and col- An interesting mechanism is the use of the collagen bind- lagen, appear to have less redundancy with other path- ing domain of GPVI as competitor for the binding of platelet ways as compared to inhibitors of secondary platelet ac- associated GPVI. Since only dimeric GPVI has a significant tivation. Little redundancy (in the absence of tissue factor affinity for collagen the therapeutic protein is expressed as a human immunoglobulin Fc domain fusion protein [161]. 1366 Current Pharmaceutical Design, 2009, Vol. 15, No. 12 Steve P. Watson

Such GPVI-Fc fusion proteins have been shown to inhibit a series of molecules (OM) partly having crossreactivity to platelet adhesion to collagen and thrombus formation under the rat protein [172]. Fab fragments of one of the generated flow. Moreover, they prevent platelet adhesion to the injured antibodies, OM4, effectively inhibited thrombus formation in vessel and thrombus formation in vivo (endothelial denuda- rats (modified Folts model of cyclic flow reduction in carotid tion of mouse carotid artery by vigorous ligation) [162]. In artery). Importantly, the Fab fragment did not induce throm- contrast, Gruner et al. showed in the same in vivo thrombosis bocytopenia, GPVI depletion nor did treatment with OM4 model an only partial inhibition at high concentration of the prolong the bleeding [175]. This clearly supports the view, fusion protein, while antibodies against GPVI were highly that anti-GPVI treatment in a competitive manner can be a inhibitory [163]. The reason for this discrepancy is unclear promising antithrombotic mechanism, with an excellent but may be based on slight but significant differences in the benefit - safety window. execution of the animal model. Support for the use of GPVI- Fc as therapeutic protein comes also from a study showing 21 that local delivery of the protein to the site of an injured ca- As the first receptor on platelets mediating firm adhesion rotid artery (ballon injury in rabbits) is able to substantially to collagen, this integrin is also a potential target for phar- attenuate thrombus formation [164,165]. This directed deliv- maceutical intervention. Derivatives of specific 21 inhib- ery is an interesting option since it ensures high local con- iting snake venom components like rhodocetin might serve centrations of the fusion protein plus it contains the anti- as starting points for such antagonists [176]. There are no thrombotic effect to the targeted site, which should be fa- reports of clinical trials with direct-acting 21 antagonists. vourable in terms of bleeding. The clinical development of Eisai is investigating an 21-expression inhibitor, E7820, GPVI-Fc (PR-15) is pursued by Trigen, Munich, Germany. for cancer indications in Phase II because of the role of the A Phase I study has been successfully finalized [166]. integrin in angiogenesis [177]. Small molecule antagonists of 2 1 for cancer and thrombosis have been described [178] GPVI (Biotie website) but further development has not been re- The crystal structure of the GPVI collagen binding do- ported. main has been recently described, which may open some avenues for the identification of small molecule inhibitors VWF [167]. However, based on the large interaction surface be- As the interaction partner in the first step of platelet ad- tween GPVI and collagen the use of biotherapeutic proteins hesion, VWF is a strong target for consideration of pharma- (antibodies or fragments thereof) seems to be the most prom- cological intervention. Targeting VWF would also have the ising alternative to find potent GPVI inhibitors. Furthermore, advantage of avoiding complications based on potential side- anti GPVI antibodies are able to induce receptor down- effects through targeting of GPIb-IX-V that might lead to regulation, which may reflect an additional anti-thrombotic thrombocytopenia given the critical role of GPIb-IX-V in mechanism. It has been shown in vivo that mice, treated with platelet formation. As discussed above the binding region of anti-GPVI antibodies are rendered GPVI-deficient and have VWF for the GPIb-receptor complex on platelets becomes significant antithrombotic characteristics in several thrombo- only available under conditions of high shear. sis models [168]. This effect of anti-GPVI antibodies nicely explains much earlier observations in patients with autoanti- AJW200 is a humanized anti-VWF-A1 antibody that has bodies against the collagen receptor. These patients also been reported to be well tolerated in a clinical Phase I study. showed a defect in collagen-induced platelet activation and This antibody had been shown to inhibit shear-induced plate- GPVI deficiency on their platelets [169]. However, the asso- let aggregation and to reduce thrombosis in coronary arteries ciation of antibody-induced GPVI down-regulation with par- in a canine coronary artery model [179,180]. Further, it tial platelet activation and transient thrombocytopenia has showed a favorable effect - bleeding ratio versus the com- been described, which makes this approach futile for clinical parator, abciximab (IIb3 inhibitor), suggesting a safer use [168]. Thus, more basic research in the principles of pharmacological profile in dogs. GPVI downregulation is warranted. Nevertheless, a recently The two most advanced approaches in targeting VWF in published elucidation of the signalling pathways involved in clinical testing are an aptamer, a protein-binding oligonu- GPVI downregulation and the description of an antibody cleotide, as well as a nanobody, a “heavy-chain only” anti- which seems to cause a decrease in surface expression of the body derived from camelids (for review see [181,182]). receptor without obvious signs of platelet activation or thrombocytopenia may open new avenues for the clinical use Archemix is investigating ARC1779, a pegylated ap- of this mechanism [170,171]. tamer binding to the VWF-A1 domain [183]. In a Phase I study in healthy volunteers it was well tolerated, with an Another option is the classical competitive inhibition of apparent elimination half-life time of 2 hours and no bleed- the collagen binding site of GPVI. To avoid any platelet ac- ing episodes, while inhibiting platelet function dose- tivation, monovalent proteins such as Fab fragments are the dependently. ARC1779 is currently in Phase II clinical de- molecules of choice. A number of antibodies against human velopment, assessing the safety, pharmacokinetics and GPVI have been generated by several groups from academia pharmacodynamics in patients with VWF-mediated platelet and industry [171-174]. Characterization of these molecules function disorders, including TTP (Thrombotic thrombocy- in vivo has been often hampered by the very narrow cross- topenia purpura), as well as acute coronary syndrome pa- reactivity of anti-human GPVI antibodies generated in mice. tients undergoing PCI (Archemix website). Since TTP is To circumvent this, Matsumoto et al. used GPVI-knock out associated with an increased plasma concentration of ultra- mice for the generation of anti GPVI antibodies, resulting in Platelet Activation by Extracellular Matrix Proteins Current Pharmaceutical Design, 2009, Vol. 15, No. 12 1367 large, highly prothrombotic multimers of VWF, a direct in- CONCLUSIONS hibition of VWF seems to be here particularly promising. The subendothelial extracellular matrix has been shown However, clinical studies will show if these predictions hold to be a powerful activator of platelets. Moreover, the inter- true. play between components of the subendothelial matrix and This is also the case for Ablynx’ ALX-0081, an anti- specific platelet receptors causes a stable adhesion of acti- VWF nanobody directed against VWF. The data from ani- vated platelets to the exposed subendothelium, which initi- mal studies in a modified Folts-model in baboons showed a ates the formation of a haemostatic plug but also localizes more pronounced effect on inhibition of thrombus formation the haemostatic reaction to the site of injury. These proper- than a combination of aspirin, heparin and clopidogrel, with ties underline a critical, life saving role of the subendothelial less bleeding complications [184]. The half-life of ALX- matrix in haemostasis. However, pathological changes 0081 in baboons was similar to the half-life of its target, within the vessel wall which include a remodelling of the VWF-A1 (8 hours). A Phase I study in healthy volunteers subendothelial matrix and a dysfunctional endothelial layer has been successfully concluded and confirmed that ALX- give rise to atherosclerosis and life threatening thrombotic 0081 adopts at least the half-life of it’s target. The nanobody complications. Besides intensive medical treatment athero- showed no immungenicity, was safe and well tolerated at all thrombosis is still a leading cause of death worldwide. doses tested, and no serious adverse events were reported. Therefore, the development of new antithrombotic and The anti-thrombotic effect was dose-dependent in a range anti-atherosclerotic mechanisms is needed. Because of the from 2 - 12 mg. A Phase Ib study in patients with stable an- importance of the subendothelial matrix for these processes gina undergoing PCI is ongoing. Phase II is scheduled to several components represent interesting drug targets. Plate- start by the end of 2008 (Ablynx website). lets interact with vessel wall associated laminin, fibronectin, vitronectin and collagen. Of these, collagen and associated GPIb VWF seem, however, of particular importance for platelet The receptor for VWF-A1 on platelets, the GPIb-V-IX activation and adhesion. Thus, blocking the interaction of complex, is another option for anti-thrombotic intervention. platelets with collagen (via its receptors GPVI, 21 or col- Besides VWF it also interacts with TSP, P-selectin, throm- lagen binding peptides) and VWF (GPIb inhibitors or mole- bin, Mac-1, high-molecular weight kininogen, FXI and FXII cules against VWF) seems to represent the most promising (reviewed in [185]). All these interactions may play a role mechanism. Several approaches have been initiated and the under pathophysiological conditions, while their relative first molecules have entered early clinical testing. The very importance is still under discussion. The main concern in promising investigational and (pre)clinical data raise hopes targeting GPIb is thrombocytopenia. Thus it will be crucial that these approaches may significantly improve the treat- to monitor this potentially life-threatening condition closely ment of atherothrombotic diseases. while investigating GPIb inhibitory principles. For in vivo ACKNOWLEDGEMENTS studies, Fab-fragments of anti-GPIb antibodies where successfully used, without causing thrombocytopenia. One ex- The research in the group of SPW is supported by the ample is the humanized anti GPIb antibody 6B4, which was British Heart Foundation, MRC and Wellcome Trust. SPW applied in a model of arterial thrombosis (modified Folts holds a BHF Chair. I thank Gemma Fuller for help with the model) in baboons [186]. While the bolus injection of 0.6 figure. I would like to express considerable gratitude for the mg/kg or 2 mg/kg 6B4 Fab fragments led to a significant many hours of helpful discussion to Drs Peter Wonerow and anti-thrombotic effect, platelet count and bleeding time were Horst Blum. not significantly changed. 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