The selectins are a well-conserved family of glycopro-tein, transmembrane molecules expressed on the surface of leukocytes, platelets, and endothelial cells. They play a crucial role in leukocyte and platelet rolling and adhesion to areas of vascular injury and inflammation.21 Three selectins have been described: L-selectin, E-selectin, and P-selectin. P-selectins are stored as transmembrane proteins in preformed cytoplasmic granules mobilized by activated platelets and endothelial cells. E-selectin is inducible on vascular endothelium, and L-selectins are expressed by nearly all leukocytes.
The selectin family has a unique extracellular structure consisting of an amino terminal calcium dependent lectin domain, an epidermal growth factor-like domain, and two to nine consensus repeat sequences (CRS) that are homologous to complement binding domains. They also possess a lipophilic transmembrane domain and a short cytoplasmic tail. The variable structural length of consensus repeat sequences determines which complementary ligand carbohydrates the conserved lectin and epidermal growth factor domains it will interact with.22 L-selectin has two consensus repeat sequences, E-selectin six consensus repeat sequences and P-selectin nine consensus repeat sequences.
Leukocyte adhesion to endothelium is controlled by the binding of vascular selectins to leukocyte expressed gly-coproteins or glycolipids containing the tetrasaccharide sialyl-Lewisx (sialic acid, galactose, fucose and N-acetyl-galactosamine).22 The ligands for P-selectin (PSGL-1) and E-selectin (not well defined) are found on leukocytes, and the ligands for L-selectin (CD34, MAdCAM-1) are found on endothelial cells. Each ligand functions by presenting O-linked saccharide chains to the calcium dependent lectin domain on the corresponding selectin. Our research has focused on modulating the P-selectin:PSGL-1 signaling axis with the therapeutic goals of preventing DVT formation, treating established DVTs and preventing chronic venous insufficiency.
P-selectin is stored in the alpha granules of platelets and in the Weibel-Palade bodies of endothelial cells (EC).23 Exposure to an activating stimulus such as thrombin results in rapid translocation of P-selectin to the cell surface, avoiding the need for transcription or translation.24 E-selectin is upregulated after the initiation of thrombosis in a transcription-dependent fashion. P-selectin can be secreted into the circulation as a component of EC and platelet-derived microparticles (MP) or, in small quantities, as a free, alternatively spliced version lacking a transmembrane domain.25 These two forms of soluble P-selectin are elevated in humans in association with atherosclerosis and thrombosis and are predictive of future adverse cardiovascular events, including myocardial infarction and stroke.26-28 Soluble P-selectin levels are also elevated at times of overwhelming systemic thrombosis and consumption, such as disseminated intravascular coagulation and heparin-induced thrombocytopenia.29
P-selectin plays a critical role in the initial adhesion and rolling of platelets and leukocytes to areas of injury and inflammation via the P-selectin receptor, P-selectin glycoprotein ligand-1 (PSGL-1). P-selectin also plays a prominent role in hemostasis and thrombosis through PSGL-1 signaling in leukocytes and platelets and GPIba in platelets. P-selectin deficient mice demonstrate a hemophilic phenotype with marked bleeding tendencies.30 Mice with a deletion of the transmembrane domain of P-selectin have elevated levels of circulating P-selectin resulting in thrombophilia. P-selectin based therapy has been demonstrated to correct a mouse model of hemophilia.31
In animal models of DVT, we, and others, have demonstrated that P-selectin expression regulates fibrin deposition and thrombus size.32,33 We have also demonstrated that P-selectin and E-selectin deletions are associated with decreased thrombosis and that the thrombi have decreased fibrin content.34 In a primate model of stasis-induced DVT, P-selectin blocking antibodies or antibodies blocking the P-selectin receptor, PSGL-1, inhibit thrombosis and promote recanalization.35,36 In animal models we have found that P-selectin blockade with monoclonal antibodies has been found to be as effective as low molecular weight heparin in promoting thrombus resolution and preventing vein wall thrombosis, all without the risk of hemorrhagic complications.37
We have also demonstrated that P-selectin inhibition is an effective treatment for established primate and rodent iliofemoral DVT through augmentation of fibrinolytic activity.38 The findings of an improvement in spontaneous thrombolysis in animals in which P-selectin is inhibited by rPSGL-Ig are similar to results found in primate, porcine, and rat models of arterial and venous thrombosis using P-selectin inhibition.36,39-41 A reduction in the fibrin content of thrombi formed in the presence of P-selectin inhibition is likely contributory, as leukocyte-platelet interactions leading to fibrin deposition are P-selectin dependent.33
Microparticles (MP) are small (less than 1 micrometer, about the size of a bacterium) phospholipid vesicles that are shed from a variety of cell types including platelets, leukocytes, and endothelial cells.42-44 Microparticles are a normal constituent of blood and can be isolated from plasma by ultracentrifugation. Microparticles lack DNA and RNA but are protein rich. All circulating blood cells, platelets, and endothelial cells are capable of releasing MP in a calcium-dependent fashion. The protein expression profile of MP depends on the mother cell of origin and the conditions influencing their production. The protein and phospholipid content of MP determines the biologic activity of MP with regard to hemostasis and other physiologic processes. Subpopulations of MP rich in tissue factor (TF) and phosphati-dylserine, two critical components of the coagulation cascade, have been identified.45,46 Several circulating markers of inflammation once thought to be soluble actually are carried by MP.47
Lipid rafts are sphingolipid ordered, cholesterol-rich microdomains floating within the more fluid cell surface bilayer (the "fluid mosaic").48 These rafts allow for the concentration of receptor clusters, G-proteins, adaptor proteins, and downstream kinases to facilitate signal transduction through the concentration of the proximal machinery of cell signaling pathways.49 Apoptotic blebs and microparticles are derived from raft-rich regions of the plasma membrane, resulting in high concentrations of cholesterol, negatively charged phospholipids, and protein complexes, as compared with the cell of origin.
Microparticles are rich in the external leaflet aminophos-pholipids phosphatidylethanolamine and phosphatidylserine providing a critical, negatively charged substrate for coagulation. Under quiescent conditions, membrane asymmetry is maintained by the active transport of nonthrombotic, choline phospholipids to the outer leaflet in exchange for amino-phospholipids by the enzymes flippase and floppase. Cellular activation results in the induction of scramblase activity and the inhibition of flippase. This results in a loss of asymmetry and induction of a procoagulant phenotype.50 Inability to mobilize aminophospholipids to the outer membrane leaflet due to defective phospholipid translocation results in a bleeding tendency manifested as Scott syndrome.51
Rafts and raft-derived MP can concentrate tissue factor (TF) in cavaolae where it is stored with tissue factor pathway
FIGURE 37.1 The role of microparticles in thrombus amplification (Adapted from Myers et al61).
FIGURE 37.1 The role of microparticles in thrombus amplification (Adapted from Myers et al61).
? microparticles in thrombus amp
? microparticles in thrombus amp inhibitor (TFPI).52 Fusion of MP with activated platelets results in decryption of TF and the initiation of thrombosis.53 Monocytes concentrate TF and PSGL-1 in rafts. Monocyte-derived MP deliver TF to areas of injury and inflammation by binding to P-selectin mobilized to the surface of activated platelets and EC, resulting in the generation of fibrin (see Figure 37.1).45 Through the depletion of cholesterol in the raft regions of platelets and leukocytes, statin drugs may decrease the risk of thrombosis through raft architectural disruption. Cholesterol-depleted platelets and leukocytes are less adherent and may release less thrombotic MP.54
The P-selectin receptor, PSGL-1, is expressed on leukocytes and platelets as well as on their derived microparticles. MP coexpressing TF and leukocyte markers have been shown to accumulate in growing thrombi in a PSGL-1: P-selectin dependent fashion.55,56 P-selectin:PSGL-1 interactions also stimulate the production of thrombogenic MP from leukocytes, particularly monocytes.31,57 These pro-thrombotic MP express TF and possess a phosphatidylserine rich anionic surface capable of assembling prothrombinase, tenase, and factor V/Va.58 Hrachinova et al. treated mice with hemophilia A with P-sel-Ig to generate prothrombotic MP. This normalized tail bleeding times and systemic coagulation times through augmentation of the extrinsic coagulation pathway.31 This strategy may result in new therapies for hemophilia A patients with alloantibodies. Human pericar-dial MP expressing TF have been demonstrated to increase thrombosis in a rat venous stasis model.59 Venous stasis and ischemia results in the upregulation of vascular P-selectin, which localizes prothrombotic MP to the area of stasis and promotes DVT formation (see Figure 37.1).60-62
In some vascular models of injury, P-selectin dependent TF accumulation and fibrin deposition begins within the first 20 seconds of injury, before leukocyte rolling occurs, suggesting that a major function of selectins in venous thrombogenesis is independent of leukocyte rolling and extravasation. P-selectin:PSGL-1 interactions are critical for the localization of prothrombotic MP to areas of injury and inflammation.63,64 The relative importance of MP-born TF in thrombosis appears to vary according to the nature and scope of the injury. MP appear to play a critical role in stasis-induced thrombosis and small vascular injuries. Chou et al. demonstrated that leukocyte-derived TF contributed significantly to thrombus formation after laser injury of the microvessels in the cremaster muscle, a small injury model. Day et al. demonstrated that leukocyte-derived TF did not contribute significantly to thrombosis following treatment of the carotid artery with ferric chloride, a dramatic, somewhat artificial and large-scale injury. It is likely that MP-bearing tissue factor contribute to the initial thrombus formation and amplification that occurs in the minutes following small scale vascular injuries (see Figure 37.2).62
Microparticles are not only prothrombotic but also appear to inhibit fibrinolysis. Platelet activator inhibitor-1 (PAI-1) is stored in the a-granules of quiescent platelets.65 PAI-1 is a potent inhibitor of tissue plasminogen activator (tPA) and urokinase-type plasminogen activators, which are largely responsible for the initiation of fibrinloysis.66 Upon activation, MP shed from platelets express PAI-1, and these MP are localized to the growing thrombus via P-selectin-PSGL-1 interactions. In this manner, platelet microparticles are not
only prothrombotic but also inhibit fibrinolysis, delaying thrombus resolution.67
Traditional anticoagulation is contraindicated in many patients and results in significant hemorrhagic complications. Patients on optimal therapy have a 20% incidence of thrombus recurrence or extension. The new, inflammatory perspective on VTE has opened up a line of attack along the axis of selectin biology. Presently, we are conducting rodent and primate research on the prophylactic and therapeutic use of compounds that inhibit P-selectin or its receptor, PSGL-1, and that can be delivered by multiple routes for use in VTE.
D-dimers are a plasmin breakdown product of insoluble, cross-linked fibrin. Although D-dimers are elevated in patients with VTE, this finding has a specificity of only 50 to 70%. In current practice D-dimer is useful only when negative in association with patients with low clinical assessment scores.68
The abundance of evidence supporting an important role for P-selectin and MP in thrombosis led us to hypothesize that they may be reliable clinical indicators of an ongoing, thrombotic event. We have found that a panel of blood tests including D-dimer, P-selectin, and MP levels is more sensi tive and specific for the detection of DVT than D-dimer alone, though still less sensitive than duplex ultrasound (US). This panel would be most useful in locations or at times when duplex US is unavailable. As our knowledge of microparticle subtypes and protein expression improves, it is likely that even more definitive tests for the detection of thrombotic events and assessment of overall cardiovascular risk will become available.
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