Extracellular matrix proteins in hemostasis and thrombosis

Abstract

The adhesion and aggregation of platelets during hemostasis and thrombosis represents one of the best-understood examples of cell-matrix adhesion. Platelets are exposed to a wide variety of extracellular matrix (ECM) proteins once blood vessels are damaged and basement membranes and interstitial ECM are exposed. Platelet adhesion to these ECM proteins involves ECM receptors familiar in other contexts, such as integrins. The major platelet-specific integrin, αIIbβ3, is the best-understood ECM receptor and exhibits the most tightly regulated switch between inactive and active states. Once activated, αIIbβ3 binds many different ECM proteins, including fibrinogen, its major ligand. In addition to αIIbβ3, there are other integrins expressed at lower levels on platelets and responsible for adhesion to additional ECM proteins. There are also some important nonintegrin ECM receptors, GPIb-V-IX and GPVI, which are specific to platelets. These receptors play major roles in platelet adhesion and in the activation of the integrins and of other platelet responses, such as cytoskeletal organization and exocytosis of additional ECM ligands and autoactivators of the platelets.

Figure 1.

ECM protein sources for hemostasis. Platelets have access to a wide variety of ECM proteins with which to interact. Multiple ECM proteins circulate in plasma, the most important for hemostasis being VWF and fibrinogen. Platelets themselves have multiple ECM proteins stored in secretory α granules, which release their contents on platelet activation; some of these secreted platelet-ECM proteins are released as soluble proteins, others are retained at the surfaces of the activated platelets, often by interactions with platelet adhesion receptors. The lumenal surfaces of endothelial cells (green) are free of ECM, but the basement membrane beneath them (gray) contains the usual basement membrane proteins plus VWF. Basement membranes of some vessels also contain additional ECM proteins. The layers below the basement membrane vary with vessel type; shown here are a smooth muscle layer (red) containing elastin, microfibrils, collagens, and fibronectin, and the surrounding connective tissue adventitial layer (black), which contains fibrillar collagens, fibronectin, and other ECM proteins. The interstitium around blood vessels (gray mesh) obviously varies with the tissue but typically contains fibrillar collagens, fibronectin, and diverse other ECM proteins. Therefore, the ECM proteins available to mediate platelet adhesion vary greatly depending on the vessels involved and on the severity of the injury (e.g., whether or not ECM below the basement membrane is exposed). The ECM also evolves considerably during the course of a hemostatic or thrombotic event. Circulating VWF can be bound directly by platelets or can bind to exposed collagen fibers. Platelet activation, either by soluble factors such as thrombin, ADP, and epinephrine or by ECM proteins such as VWF, collagens, and laminins, releases additional ECM proteins and activates receptors that can recruit additional ECM proteins such as fibrinogen. Platelets have receptors for many, maybe all, of these ECM proteins and a reasonable hypothesis is that they have evolved to adhere to whatever ECM proteins they encounter, no matter where the lesion occurs or how severe the injury. ECM proteins known to have major roles in platelet adhesion and aggregation are bolded.

Figure 2.

von Willebrand factor. (A) Domain structure and binding sites of von Willebrand factor (VWF). The standard domains (as per the SMART protein domain site http://smart.embl-heidelberg.de) are shown in the diagram. In the hemostasis field, VWF is traditionally designated with A, B, C, and D repeats. The A repeats correspond with VWA domains and each D repeat corresponds with a combination of tandem VWD/C8/TIL domains. The carboxyl terminus of the molecule comprises three VWC domains and a carboxy-terminal knot domain. These do not correspond exactly with the B and C repeats. The C8, TIL, VWC, and CT domains are all cysteine-rich, making the drawing of boundaries between domains nontrivial; here we have used the standard domains based on hidden Markov models and comparisons with other proteins. Binding sites are shown for the coagulation cofactor, factor VIII (D3 repeat), for collagen (collagen binds the A3 domain) and for the two major receptors for VWF, the GPIb-V-IX complex (A1 domain) and integrin αIIbβ3 (an RGD site between the last two VWC domains; see text). During biosynthesis and processing, the prodomain comprising D1 and D2 repeats is separated from the mature VWF by cleavage after a dibasic amino acid pair by the enzyme PACE (paired basic amino acid cleaving enzyme). VWF monomers then associate into dimers (∼500 kDa) through disulfide bonding of their carboxy-terminal domains. (B) VWF multimerization. The tail–tail VWF dimers then associate by amino-terminal disulfide bonding into ultralarge VWF polymers (>10 million Da; >40 monomers) that are stored in Weibel-Palade bodies. When these are released, they form enormous extended fibrils, which extend under the shear forces in the circulation and are cleaved by ADAMTS13 to smaller but still large multimers containing fewer than 40 monomers. ADAMTS13 cleaves in the A2 VWA domain (see text). (C) Domain key shows the domains making up VWF and other features such as intersubunit S—S bonds and the integrin-binding RGD site.

Figure 3.

Platelet receptors for ECM proteins. (A) The GPIb-V-IX complex—the major receptor for von Willebrand factor (VWF), which binds through its A1 VWA domain (blue pentagon) to the LRR repeat region of GPIbα. The receptor is constitutively active and has nine subunits: two copies of GPIbα, each of which associates with two copies of GPIbβ and one of GPIX—the two GPIb-IX heterotetramers are associated with a single copy of GPV. The cytoplasmic domains of GPIbα and GPIbβ bind filamin and a number of signal transduction proteins, including Src (red sphere), 14-3-3 (green), and calmodulin (yellow), and activate PI3-kinase and phospholipase Cγ2. (B) GPVI, a receptor for fibrillar collagens, has two Ig domains and is noncovalently associated with the Fc receptor-γ (FcRγ) chain, which acts as a signal transduction subunit. On ligand binding, a Src family kinase (red sphere) phosphorylates two tyrosine residues in the immunoreceptor tyrosine-based activation motif (ITAM) motif within the FcRγ chain. Syk kinase (orange) binds the phosphorylated ITAM motif and activates phospholipase Cγ2. Ca²⁺ generated downstream from IP3 released by PLC stimulates the Rap-GEF, CalDAG-GEF1, to activate Rap1, which leads to association of talin-1 with integrins, thereby activating them. Diacylglycerol (DAG) also induces PKC-mediated release of granule contents, including soluble agonists and ECM components. (C) Integrins are activated by the VWF and collagen receptors (or by GPCRs bound by other platelet agonists) through binding of talin-1 and kindlin-3 to their β subunit cytoplasmic domains. Once activated, integrins can bind their ECM protein ligands. Several β1 integrins are expressed on platelets at a few thousand copies per platelet, most notably α2β1 and α6β1, receptors for collagens and laminins, respectively, but also α5β1, a receptor for fibronectin and fibrillin and αvβ3, which binds many ECM proteins (see text). The major platelet integrin, αIIbβ3, is present at 60–80,000 copies per platelet and is the major receptor mediating platelet aggregation through its binding to fibrinogen (and also to fibronectin, VWF, and several other ECM proteins—see text). Most integrins bind their ligands (black) through a binding site at the junction between the head domains of the two subunits—a β propellor domain in the α subunit (dark blue) and a VWA domain in the β subunit (red). In contrast, α2β1 binds through an additional VWA domain inserted into the β propellor domain (not shown).