The Gplb-IX-V Receptor Complex
The platelet Gp Ib-IX-V complex—universally credited as an essential component of hemostasis and as a notorious mediator of arterial atherothrombosis—is also a critically important factor in the pathogenesis of human diseases affecting the microvasculature. It plays a primary role in afferent arteriolar and capillary thrombosis in thrombotic thrombocytopenic purpura (TTP) and the haemolytic-uremic syndrome (HUS). It may also play a primary role in platelet adherence to inflamed venular endothelium and a significant secondary role in effecting venular thrombosis after leukocytes have been recruited to the injured microvasculature.
GpIb-IX-V directs specific platelet responses by organizing disparate factors operating within Virchow's triad into a single series of functional responses: adhesion, secretion, and aggregation. Its functional versatility—as a switch for turning platelet-dependent hemostasis and thrombosis "on" within different vascular and rheological microenviron-ments—relates to its primary structural attributes. GpIb is made up of GpIba and GpIbb disulfide bonded at a single perimembranous extracellular site. There are about 25,000 GpIb heterodimers per platelet noncovalently associated with GpIX and GpV, and the stoichiometry of the GpIba/p-IX-V complex is believed to be 2:2:2:1. Each of the members of the GpIb-IX-V complex is a related member of the leucine-rich repeat protein family and each has a single transmembranous domain. The "business ends" of the complex are found mainly on GpIba: the large extracellular N-terminal ligand binding domain and the 96 amino acid cytoplasmic C-terminal cytoskeletal-binding and signaling domain. In addition, experimental data suggest that the 34 amino acid cytoplasmic domain of GpIbb modulates platelet activation and the extracellular domain of GpV, which is a substrate for thrombin cleavage, modulates platelet responses to thrombin.
Pathological shear stress-dependent platelet adherence in arteries and arterioles is triggered by platelet GpIba binding to plasma or vessel wall von Willebrand factor (VWF). VWF is synthesized by vascular endothelium and by megakaryocytes. It is constitutively released adluminally (into the subendothelium) and abluminally (into the blood) by the endothelium, and it is also stored and secreted following cellular activation (stored in endothelial cell Weibel-Palade bodies and in platelets' a-granules). Subendothelial VWF is most abundant in the macrovasculature large veins and arteries (most large veins > pulmonary artery > cerebral arteries > aorta > coronary arteries > renal arteries > hepatic arteries > pulmonary vein). It is generally less abundant in microvasculature subendothelium, and its distribution in the microvasculature is noteworthy: venules > arterioles > capillaries, with very little or no VWF observed in any embryo capillary bed (in mice) and with relatively little VWF observed in adult myocardium (in pigs).
It is a multimeric protein built up of tens to hundreds of disulfide-bonded multivalent protomeric units. Larger multimers appear to have greater hemostatic and prothrombotic properties. Each protomeric subunit is composed of two disulfide-linked mature VWF polypeptides, each one of which is divided structurally and functionally into several domains: the A, B, C, and D domains. The A domains are of particular importance because the A1 domain forms the primary GpIba recognition site and binds to type VI collagen, the A2 domain contains the recognition sequence for degradation by the VWF multimer-cleaving protease ADAMTS (a disintegrin and metalloproteinase with thrombospondin type 1 motif) 13, and the A3 domain binds to fibrillar collagens type I and III found in arterial subendothelium, thus allowing soluble VWF to tack down onto the subendothe-lium of ruptured atherosclerotic plaques. Two other domains of VWF are important for hemostasis and thrombosis: The C2 domain contains a RGD integrin recognition domain essential for VWF binding to platelet aIIbp3 and an N-terminal D domain binds Factor VIII.
VWF binding to GpIba is required for microvascular hemostasis, which appears to be triggered in the injured arteriole—a high shear stress microenvironment—by soluble and subendothelial VWF alone or bound to collagen (type VI may be the predominant microvascular collagen) attaching to platelets via GpIba, with the subsequent activation of aIIbb3 to a ligand-receptive conformation. The high shear stress in the arteriole limits fibrin deposition and leukocyte recruitment, and neither soluble coagulation factors nor leukocyte number or function contribute in any clinically important manner to microvascular hemosta-
sis in the epithelium (such as skin, mucous membranes, and the urinary and GI tracts). As shear stress falls in the occlusive hemostatic plug to venular levels, fibrinogen binding to activated aIIbp3 is important for interplatelet cohesion. The importance of fibrinogen is emphasized by clinical observations that fibrinogen deficiency causes a severe hemostatic defect. Regional regulation of the VWF-triggered hemostatic plug induced by a bleeding time wound is remarkably fine-tuned, as the platelet-rich thrombi accrue only at the mouths of the transected arterioles, and little or no platelet accumulation occurs within the adjacent arteriole lumen.
VWF-dependent arteriolar platelet thrombosis is the hallmark of TTP and HUS. In TTP the primary pathogenetic event may be an acquired deficiency of ADAMTS13, resulting in persistent "ultralarge" VWF multimers—on endothelial cell (EC) surfaces and in the blood—effecting GpIb-IX-V-dependent platelet adhesion, secretion (predominantly dense granules which contain costimulatory adeno-sine diphosphate), aIIbp3 activation, and aggregation. In diarrhea-associated HUS the primary pathophysiology may involve an enterotoxin-mediated overstimulation of "ultralarge" VWF multimers from arteriolar endothelial Weibel-Palade bodies. This bolus of large VWF multimers somehow remains attached to the EC surface and thereby recruits passing platelets through the shear-dependent binding of platelet GpIba to the EC-associated VWF. A similar effect may occur in low-shear-stress postcapillary venules when rapid EC Weibel-Palade body release is stimulated by calcium ionophore or histamine, suggesting that there may be clinical conditions associated with venular inflammation in which part of the pathophysiological process is caused by VWF/GpIb-IX-V mediated platelet recruitment within a low shear stress microenvironment.
There are several other molecules relevant to micro-vascular hemostasis and thrombosis that bind directly to GpIb-IX-V. These include P-selectin, which is expressed on inflamed venules and mediates platelet rolling through a direct interaction with GpIba. Platelet GpIba binding to the leukocyte integrin receptor Mac-1—expressed on activated neutrophils and macrophages—is involved in leukocyte recruitment to platelet thrombi and in platelet recruitment to sites of microvascular leukocyte accumulation. Platelet GpIba binds directly to thrombin and Factor XI, and such binding appears to accelerate coagulation at the surface of activated platelets under low-shear-stress (capillary and venular) conditions. GpIba also binds to high-molecular-weight kininogen and Factor XII, and these interactions block thrombin binding to GpIb-IX-V and thrombin-induced platelet activation. Thrombin also binds to GpV and this interaction may be involved in attenuating throm-bin-induced platelet activation.
Although the GpIb-IX-V complex is required for the initiation of microvascular hemostasis and is a primary factor contributing to microvascular thrombosis in TTP and HUS, several additional platelet receptors are also involved in both microvascular physiology and pathology. In every case, the co-receptor functions to support platelet-mediated microvascular responses after GpIb-IX-V engages ligands (the co-receptor supports postadhesion responses), and in some cases the co-receptor requires activation or expression downstream of VWF/GpIb-IX-V interactions (aIIbp3 and aip2 are "activated," while P-selectin is expressed following a-granule secretion). Observations of human and/or mouse deficiencies of aIIbft3 (the human defect is called Glanzmann's thrombasthenia) reveal that it causes a severe bleeding disorder but protects against macrovascular arterial thrombosis. Similar observations have been made with the protease-activated receptors that mediate thrombin-induced platelet activation, the thromboxane A2 receptor, and the P2Y12 receptor that binds adenosine diphosphate (ADP), is the target of thienopyridine antiplatelet drugs, and appears to be indispensable to platelet aggregation triggered by VWF binding to GpIb-IX-V. Similar but lesser effects on hemostasis and macrovascular thrombosis are seen in mice lacking the P2Y1 receptor or P-selectin. In contrast, human deficiencies of the collagen receptors—GpVI or integrin a2ft1—result in a mild bleeding diathesis, while GpVI- or a2pi-deficient mice have only a small or no hemostatic defect, respectively. Either GpVI or a2pi deficiency or pharmacological perturbation results in delayed and decreased ex vivo thrombus formation on type 1 collagen under both arteriolar and low-shear-stress conditions, indicating that GpVI and a2pi (probably to a lesser extent in comparison to GpVI) are important but secondary mediators of microvascular thrombosis. The prototypical example of a platelet co-receptor that appears to effect hemostasis and thrombosis paradoxically is platelet endothelial cell adhesion molecule (PECAM)-l, which is expressed by the microvascular endothelium and platelets. Mice deficient in PECAM-1 have a hemostatic defect not because of a loss of platelet expression, but because they lack homotypic interactions between PECAM-1 on adjacent ECs needed to regulate the endothelial component of Virchow's hemostatic response. In fact, PECAM-1-deficient platelets are actually hyperresponsive to both VWF and collagen because PECAM-1 is a negative regulator of GpIb-IX-V- and GpVI-dependent signaling.
ADAMTS13 is the VWF multimer-cleaving protease. It is synthesized in the liver, circulates in blood, and may attach to the vascular endothelium. Although the regional distribution of ADAMTS13 activity is not yet understood, based on the end-organ pathology of TTP it appears to play an important role in processing "ultralarge" VWF multimers—released constitutively and secreted following EC stimulation—in the cerebral, mesenteric, myocardial, splenic, renal, pancreatic, and adrenal arterioles. It also appears that arteriole-level shear stress is required for ADAMTS13-mediated cleavage of "ultralarge" VWF multimers: higher levels of shear stress open up or untangle the multimers and thereby expose the ADAMTS13 cleavage site in the VWF monomer A2 domain. The hypothetical mechanism by which ADAMTS13 deficiency leads to TTP presents a tidy pathophysiological model. Under physiological conditions, ADAMTS13 is synthesized, homes to the arteriole EC, and, under arteriole levels of shear stress, breaks down prothrombotic "ultralarge" VWF multimers. This maintains blood flow within the microvasculature. When ADAMTS13 is deficient, arteriolar-level shear stress triggers platelet Gplba binding to the unprocessed multimers, thus causing the arterioles to become occluded with both EC-attached platelets and platelet clumps that exceed the diameter of the narrowing arteriolar branches. This leads to ischemia and infarction of the involved organs.
An intact vascular endothelium actively maintains blood fluidity. Recall that a bleeding time wound shows platelet thrombus formation only at the site of arteriolar transection. This is because the adjacent arteriolar, capillary, and venular ECs constitutively secrete or express on their surface molecules that prevent GpIb-IX-V-dependent platelet adhesion, secretion, and aggregation. These include PGI2 and nitric oxide (which inhibit Gplba-mediated adhesion and signaling), the ectoADPase CD39 (which breaks soluble ADP down), thrombomodulin and heparin sulfates (which bind and inactivate thrombin), and urokinase-type plasminogen activator (uPA) and tissue plasminogen activator (tPA) (which generate plasmin capable of degrading VWF and fibrinogen). The capillary endothelium is particularly richly endowed with tissue factor pathway inhibitor (TFPI), which is both secreted into the blood and retained on capillary endothelium, where it binds to and inactivates the tissue factor/Factor VIIa/Factor Xa complex, thereby eliminating thrombin generation and the assembly of a platelet GpIba-based site of fibrin generation and Factor XI activation. This suggests that the capillary may be an important gate preventing the initiation of coagulation despite continuous low-level exposure to prothrombotic stimuli.
Only uPA or tPA deficiency is associated with spontaneous microvascular thromboses, but a deficiency of any one of these antiplatelet or anticoagulant molecules leads to exaggerated responses to thrombotic stimuli. The exception to this is CD39 deficiency, which causes a severe bleeding diathesis due to compromised hemostasis because elevated blood levels of adenine nucleotides lead to P2Yj-mediated platelet desensitization. This is a useful reminder that time-dependent desensitization can affect many components of Virchow's triad.
Platelet GpIb-IX-V-dependent hemostasis is mainly a physiological arteriolar response to injury. In addition to the blood factors that interact directly with Gplba (see the earlier section on Gplb-IX-V ligands), there are blood elements that modify GpIb-IX-V/vascular interactions indirectly. Perhaps most important is the red cell. Under arteriolar flow conditions the red cells flow centrally and push the platelet stream peripherally toward the arteriolar wall. Because blood flow in a tubular arteriole can be considered to be parabolic and comprised of an infinite number of infinitesimal laminae, the centripetal movement of platelets exposes them to flow laminae of lowest velocity and highest shear stress, thus slowing them and making wall collisions more efficient (or sticky). As arterioles narrow and branch into capillaries their luminal diameter narrows, red cells are excluded from the central stream (and eventually the entire stream), flow velocity falls, and platelets become evenly dispersed throughout the bloodstream. If the average pulmonary capillary diameter is calculated to be 5.8 mm, and the size of a platelet is 3 mm and a red cell is 7 mm, it becomes intuitively apparent that platelets flow relatively better (e.g., faster) than red cells through capillaries. This facilitates red cell-mediated oxygen uptake (in the pulmonary alveoli) and delivery (everywhere else). It also keeps platelets from slowing and sticking: Platelet/capillary interactions are minimized simply because fewer platelets are at the wall and more platelets are in the central stream with highest flow velocity. So platelet thrombosis in capillary beds adjacent to bleeding time wounds does not occur, at least in part, because of rheological factors—which are a direct consequence of capillary diameter and red cell size, shape, and deformability—that keep platelets flowing rapidly through the thin central stream of capillary blood (calculated velocity of platelet flow through a pulmonary capillary is about 500 mm per second).
A similar rheological environment is found in the venules: Until red cells that have squeezed their way through the capillary bed gradually queue back into concentric central stream laminae, platelets remain randomly dispersed throughout the venular lumen even as the flowing blood accelerates up to velocities three to four times greater than those found in the capillaries. This means that platelet and venule wall collisions are relatively rare and that efficient collisions (collisions resulting in attachment) between platelet GpIba and inflamed venular P-selectin or surface VWF are even rarer, suggesting that other blood factors enter the milieu and participate in pathological venular thrombosis. Perhaps the most important factor is the state of platelet activation: Platelets that enter the capillaries and venules already activated are most likely to be deposited in these vascular beds. The mechanisms by which platelet activation affects their adherence to venular endothelium are unclear, although it appears that P-selectin on activated platelets is the key and that it binds to venular and arteriolar endothelium by recognizing EC PSGL-1 and related molecules. Activated platelets also bind lymphocytes, neu-trophils, and monocytes, and they release growth factors and cytokines. These responses—alone or in combination, but always operating within Virchow's triad—contribute substantially to pathological venular thrombosis. Mechanisms by which platelets become activated within the venular circuit are not clear except in one case: In bacterially induced sepsis, splenic venular endothelium is activated to express tissue factor (TF), which generates thrombin through the "extrinsic" coagulation pathway (the TF/Factor Vila complex activates Factor X, and Factor Xa—by a calcium and phospholipid-dependent reaction—cleaves prothrombin to thrombin).
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