Extracellular Domain Of GPIbα Research Articles

Dual roles of fucoidan-GPIbα interaction in thrombosis and hemostasis: implications for drug development targeting GPIbα

BackgroundPlatelet GPIbα–von Willebrand factor (VWF) interaction initiates platelet adhesion, activation, and thrombus growth, especially under high shear conditions. Therefore, the GPIb–VWF axis has been suggested as a promising target against arterial thrombosis. The polysaccharide fucoidan has been reported to have opposing prothrombotic and antithrombotic effects; however, its binding mechanism with platelets has not been adequately studied. ObjectiveThe objective of this study was to explore the mechanism of fucoidan and its hydrolyzed products in thrombosis and hemostasis. MethodsNatural fucoidan was hydrolyzed by using hydrochloric acid and was characterized by using size-exclusion chromatography, UV-visible spectroscopy, and fluorometry techniques. The effects of natural and hydrolyzed fucoidan on platelet aggregation were examined by using platelets from wild-type, VWF and fibrinogen-deficient, GPIbα-deficient, and IL4Rα/GPIbα-transgenic and αIIb-deficient mice and from human beings. Platelet activation markers (P-selectin expression, PAC-1, and fibrinogen binding) and platelet-VWF A1 interaction were measured by using flow cytometry. GPIbα–VWF A1 interaction was evaluated by using enzyme-linked immunosorbent assay. GPIb-IX–induced signal transduction was detected by using western blot. Heparinized whole blood from healthy donors was used to test thrombus formation and growth in a perfusion chamber. ResultsWe found that GPIbα is critical for fucoidan-induced platelet activation. Fucoidan interacted with the extracellular domain of GPIbα and blocked its interaction with VWF but itself could lead to GPIbα-mediated signal transduction and, subsequently, αIIbβ3 activation and platelet aggregation. Conversely, low-molecular weight fucoidan inhibited GPIb-VWF–mediated platelet aggregation, spreading, and thrombus growth at high shear. ConclusionFucoidan-GPIbα interaction may have unique therapeutic potential against bleeding disorders in its high-molecular weight state and protection against arterial thrombosis by blocking GPIb–VWF interaction after fucoidan is hydrolyzed.

S100A8/A9 drives the formation of procoagulant platelets through GPIbα

AbstractS100A8/A9, also known as “calprotectin” or “MRP8/14,” is an alarmin primarily secreted by activated myeloid cells with antimicrobial, proinflammatory, and prothrombotic properties. Increased plasma levels of S100A8/A9 in thrombo-inflammatory diseases are associated with thrombotic complications. We assessed the presence of S100A8/A9 in the plasma and lung autopsies from patients with COVID-19 and investigated the molecular mechanism by which S100A8/A9 affects platelet function and thrombosis. S100A8/A9 plasma levels were increased in patients with COVID-19 and sustained high levels during hospitalization correlated with poor outcomes. Heterodimeric S100A8/A9 was mainly detected in neutrophils and deposited on the vessel wall in COVID-19 lung autopsies. Immobilization of S100A8/A9 with collagen accelerated the formation of a fibrin-rich network after perfusion of recalcified blood at venous shear. In vitro, platelets adhered and partially spread on S100A8/A9, leading to the formation of distinct populations of either P-selectin or phosphatidylserine (PS)-positive platelets. By using washed platelets, soluble S100A8/A9 induced PS exposure but failed to induce platelet aggregation, despite GPIIb/IIIa activation and alpha-granule secretion. We identified GPIbα as the receptor for S100A8/A9 on platelets inducing the formation of procoagulant platelets with a supporting role for CD36. The effect of S100A8/A9 on platelets was abolished by recombinant GPIbα ectodomain, platelets from a patient with Bernard-Soulier syndrome with GPIb-IX-V deficiency, and platelets from mice deficient in the extracellular domain of GPIbα. We identified the S100A8/A9-GPIbα axis as a novel targetable prothrombotic pathway inducing procoagulant platelets and fibrin formation, in particular in diseases associated with high levels of S100A8/A9, such as COVID-19.

Murine Studies to Test the Efficacy of Platelet Transfusions in the Management of Platelet Function Disorders

Background -In platelets, the nucleotide exchange factor CalDAG-GEFI (Cdg1) is critical for efficient integrin-mediated aggregation upon cellular stimulation. Mutations in Cdg1 have recently been shown to cause a novel platelet function disorder, which is characterized by partially impaired platelet aggregation and moderate-to-severe bleeding. Interestingly, platelet transfusions were successful in the treatment of bleeding in some but not all of these patients.Aims -In this study we investigated the hypotheses that (1) the hemostatic efficacy of transfused platelets is diminished by circulating malfunctional platelets, and (2) a certain ratio of “good” to “bad” platelets has to be reached in order to correct the hemostatic defect observed in these patients.Methods - A recently developed adoptive platelet transfer model was utilized to establish defined levels and ratios of wild-type (WT) to Cdg1-/- platelets in thrombocytopenic recipient mice. Additionally, WT platelets were transfused into mice lacking Cdg1, talin-1 or the extracellular domain of GPIbα (IL4α/GPIbα-tg). Hemostatic plug formation was visualized using intravital microscopy following laser ablation injury to the saphenous vein.Results - Our studies demonstrate that WT platelets are less effective in forming hemostatic plugs when co-transfused into thrombocytopenic mice with large numbers of Cdg1-/- platelets. Consistently, transfusion of WT platelets to a final level of 5 x 107 platelets/mL in circulation had only a minimal effect on the markedly prolonged bleeding time observed in Cdg1-/- or talin1-/- mice, while the same number of platelets rescued the hemostasic defect seen in platelet depleted or non-depleted IL4α/GPIbα-tg mice. Full recovery of hemostasis in Cdg1-/- mice required a final concentration of 2.5 x 108 WT platelets/mL or a ratio of not more than three Cdg1-/- platelets for every WT platelet transfused into these mice.Conclusion - Our studies in mouse models suggest limited efficacy of platelet transfusion therapy in the treatment of platelet function disorders due to competition between “good” and “bad” platelets for von Willebrand factor adhesion sites along the damaged endothelium . To correct the bleeding associated with the platelet function disorder, the fraction of transfused WT platelets has to reach ~25% of all platelets in circulation. DisclosuresNo relevant conflicts of interest to declare.

Desialylation of O-glycans on glycoprotein Ibα drives receptor signaling and platelet clearance.

During infection neuraminidase desialylates platelets and induces their rapid clearance from circulation. The underlying molecular basis, particularly the role of platelet glycoprotein (GP)Ibα therein, is not clear. Utilizing genetically altered mice, we report that the extracellular domain of GPIbα, but neither von Willebrand factor nor ADAM17 (a disintegrin and metalloprotease 17), is required for platelet clearance induced by intravenous injection of neuraminidase. Lectin binding to platelet following neuraminidase injection over time revealed that the extent of desialylation of O-glycans correlates with the decrease of platelet count in mice. Injection of α2,3-neuraminidase reduces platelet counts in wild-type but not in transgenic mice expressing only a chimeric GPIbα that misses most of its extracellular domain. Neuraminidase treatment induces unfolding of the O-glycosylated mechanosensory domain in GPIbα as monitored by single-molecule force spectroscopy, increases the exposure of the ADAM17 shedding cleavage site in the mechanosensory domain on the platelet surface, and induces ligand-independent GPIb-IX signaling in human and murine platelets. These results suggest that desialylation of O-glycans of GPIbα induces unfolding of the mechanosensory domain, subsequent GPIb-IX signaling including amplified desialylation of N-glycans, and eventually rapid platelet clearance. This new molecular mechanism of GPIbα-facilitated clearance could potentially resolve many puzzling and seemingly contradicting observations associated with clearance of desialylated or hyposialylated platelet.

The CX3C chemokine fractalkine mediates platelet adhesion via the von Willebrand receptor glycoprotein Ib

AbstractThe membrane-anchored CX3C chemokine fractalkine (FKN) is expressed on activated endothelium and is associated with the development of atherosclerosis. The potential of FKN in mediating platelet adhesion beyond platelet activation remains unexplored to date. A flow-based adhesion assay was used to study the adhesion of platelets to immobilized FKN under physiologic flow conditions. Platelet adhesion to von Willebrand factor (VWF) was increased in the presence of FKN at 600 inverse seconds. Additional platelet adhesion to FKN coimmobilized with VWF was dependent on the FKN receptor CX3CR1 and activation of glycoprotein (GP) IIb/IIIa. The number of platelets rolling on VWF was likewise enhanced in the presence of FKN. The enhancement of rolling on FKN and VWF was insensitive to anti-CX3CR1 antibody but was fully inhibited by neutralizing GPIbα function. The extracellular domain of GPIbα was covalently coupled to fluorescent microspheres, and microsphere binding was significantly higher in the presence of FKN. Platelet adhesion to activated endothelium in vitro and to intact human arteries was substantially increased in an FKN-dependent manner. These data demonstrate that endothelial expressed FKN activates platelets via its cognate receptor CX3CR1, whereas platelet adhesion is predominantly mediated by GPIbα and independent of CX3CR1.

Key role of glycoprotein Ib/V/IX and von Willebrand factor in platelet activation-dependent fibrin formation at low shear flow

AbstractA microscopic method was developed to study the role of platelets in fibrin formation. Perfusion of adhered platelets with plasma under coagulating conditions at a low shear rate (250−1) resulted in the assembly of a star-like fibrin network at the platelet surface. The focal fibrin formation on platelets was preceded by rises in cytosolic Ca2+, morphologic changes, and phosphatidylserine exposure. Fibrin formation was slightly affected by αIIbβ3 blockage, but it was greatly delayed and reduced by the following: inhibition of thrombin or platelet activation; interference in the binding of von Willebrand factor (VWF) to glycoprotein Ib/V/IX (GpIb-V-IX); plasma or blood from patients with type 1 von Willebrand disease; and plasma from mice deficient in VWF or the extracellular domain of GpIbα. In this process, the GpIb-binding A1 domain of VWF was similarly effective as full-length VWF. Prestimulation of platelets enhanced the formation of fibrin, which was abrogated by blockage of phosphatidylserine. Together, these results show that, in the presence of thrombin and low shear flow, VWF-induced activation of GpIb-V-IX triggers platelet procoagulant activity and anchorage of a star-like fibrin network. This process can be relevant in hemostasis and the manifestation of von Willebrand disease.

Binding Properties of a Human Anti-Ib ScFv Antibody (HIb-1) to Native, Recombinant, and Transgenicially Expressed Human Platelet GP Ibα Antigen.

HIb-1 is a single chain antibody (ScFv) originally obtained from the Griffin.1 library. ScFv from this library are derived from the in vitro recombination of germline human immunoglobulin VH and VL chain segments whose diversity has been greatly increased by mutagenesis directed at the CDR3 regions. HIb-1 was selected from the library by sequential biopanning first against CHO cells surface-expressing human GPIbα, and then against human platelets. Previous studies using Western blot analysis have shown that HIb-1 binds to an epitope within the 45 kDa amino-terminal portion of GPIbα (Lapan, Lyle, and Miller, 1999, Thromb. Haemost. 82S:393). HIb-1 displays inhibitory activity against both ristocetin-induced and shear-induced platelet aggregation. Biacore surface plasmon resonance analysis was used to establish dissociation constants between HIb-1and either the extracellular domain of GPIbα (i.e., “glycocalicin” or GC) derived from normal human platelets or the CHO-cell secreted GPIbα1–483 recombinant extracellular protein. With native GC immobilized on dextran sulfate, the KD for binding by analyte HIb-1 was ~600nM. With the wild-type recombinant GPIbα1–483 bound to dextran sulfate, the KD was only slightly higher at ~900 nM. We additionally studied a double mutant increase-of-function GPIbα1-483 containing the Gly233→Val233 and Met239→Val239 substitituions. The KD of HIb-1 binding to the double mutant was virtually identical to that of the wild-type, at ~900 nM. Binding of HIb-1 to native GC immobilized in an ELISA assay format was also performed. HIb-1 showed saturable binding, with a half-maximal binding concentration of approximately 170 nM. Flow cytometric analysis was also performed on platelets from murine GPIbα-null (i.e., “Bernard-Soulier”) mice that had been rescued with the human GPIbα transgene (Ware, Russell, and Ruggeri, 2000, PNAS , 97:2803), as well as upon normal mice. Murine platelets were gated using a rat anti-murine GPIIb/IIIa antibody. HIb-1 did not bind to normal murine platelets, but showed strong binding to platelets from the transgenic animals, comparable in intensity to that seen with the GPIbα mab SZ-2 (for which quantitative bead analysis indicated approximately 8,000 GPIbα receptors per platelet). It should be noted that the binding capabilities of HIb-1 to human GPIbα are those of a monovalent ScFv molecule of approximately 30 kDa, and may be sufficient for studies of in vivo GPIbα inhibition either in the case of acute injury model or of longer-term models involving repeated dosing. Model systems of more chronic inhibition, however, may require conversion of the ScFv into IgG molecules, in order to achieve greater avidity.

Filamin A Binding Stabilizes Nascent Glycoprotein Ibα Trafficking and Thereby Enhances Its Surface Expression

The glycoprotein (Gp) Ib-IX-V complex is essential for platelet-mediated hemostasis and thrombosis. The cytoplasmic domain of its largest polypeptide subunit GpIbalpha possesses a binding region for filamin A, which links GpIb-IX-V to the platelet cytoskeleton. There is evidence that filamin A binding to GpIbalpha directs the surface expression of GpIb-IX. To investigate the mechanism of this effect, we examined GpIbalpha biosynthesis in Chinese hamster ovary (CHO) cells stably co-expressing wild-type or mutant GpIbalpha with GpIbbeta, GpIX with and without filamin A. We observed that surface GpIbalpha expression is enhanced in CHO cells co-expressing human filamin A. In comparison with cells expressing only GpIbalpha, GpIbbeta, and GpIX (CHO-GpIbalpha/betaIX), lysates from CHO-GpIbalpha/betaIX + filamin A-expressing cells showed greater amounts of immature, incompletely O-glycosylated and fully mature GpIbalpha, but lesser amounts of the approximately 15-kDa C-terminal peptide released when the extracellular domain of GpIbalpha is cleaved by proteases. When filamin A binding is eliminated by truncation of GpIbalpha at C-terminal residue 557 or by a deletion between amino acids 560-570, the decreased synthesis of mature GpIbalpha is accompanied by decreased immature GpIbalpha and by an increased immunodetectable C-terminal peptide. The synthesis of mature GpIbalpha in CHO-GpIbalpha/betaIX cells is eliminated by brefeldin A (which inhibits transport out of the endoplasmic reticulum (ER)) and restored by lactacystin (which inhibits proteasomal degradation). These results suggest that GpIbalpha binds to filamin A within the ER and that filamin A binding directs post-ER trafficking of GpIbalpha to the cell surface.

GPIbα Is Essential for Platelet Adhesion during Thrombus Formation: Studies with Mutant Mice Deficient in the Extracellular Domain of GPIbα.

GPIbα binding to von Willebrand factor (vWf) exposed at a site of vascular injury is the first step in the formation of a hemostatic plug. In addition, GPIbα binding to platelet-bound vWf has previously been shown to play a key role in the incorporation of circulating platelets into a growing thrombus. However, studies in vWf-deficient mice demonstrated delayed but not absent arterial thrombus formation, suggesting that GPIbα may bind a ligand other than vWf to facilitate platelet adhesion, or that a platelet surface receptor other than GPIbα can mediate platelet adhesion in arterioles in the absence of vWf. Here we studied thrombus formation in transgenic mice expressing GPIbα in which the extracytoplasmic sequence has been replaced by an isolated domain of the human interleukin 4 receptor (IL4R-tg mice). Early platelet adhesion to ferric chloride-treated mesenteric arterioles in IL4R-tg mice was decreased by >98% when compared with controls. As a consequence, thrombus formation was completely inhibited in all of the mutant mice. To study the role of GPIbα in platelet adhesion to already adherent platelets, we studied thrombus formation in wild-type mice infused with wild-type platelets labeled with calcein-green and IL4R-tg platelets labeled with calcein-orange/red. Upon ferric chloride-induced injury, wild-type but not IL4R-tg platelets incorporated into the growing thrombus. A similar result was observed with wild-type platelets treated with O-sialoglycoprotein endopeptidase to remove the 45 kD N-terminal domain of GPIbα. In summary, our studies in GPIbα mutant mice provide additional evidence that under arterial flow conditions GPIbα is the only receptor expressed on the platelet surface that mediates initial platelet adhesion to the subendothelium as well as to already adherent platelets. Our data further suggest that a ligand other than vWf may contribute to GPIbα-dependent platelet adhesion to subendothelium and to a growing thrombus.

The Platelet That Came in From the Cold

Hoffmeister et al. discovered that clearance of chilled platelets from the blood depended upon enhanced phagocytosis by liver macrophages. Platelets, anucleate blood cells that adhere to sites of vascular injury and promote formation of blood clots, disappear from the circulation if they have been chilled, a property that limits long-term storage of platelets for transfusions. This "clearance" of prechilled platelets has been attributed to shape changes leading to entrapment in the microcirculation. Hoffmeister et al. , however, showed that chilled mouse platelets treated to maintain their shape, which appeared functionally normal, still disappeared. The authors used labeled platelets injected into mice to show that chilled platelets disappeared more rapidly than platelets stored at room temperature and that they accumulated in the liver. Chilled platelets adhered to Kupfer cells (liver macrophages); both rapid clearance and adherence to macrophages depended on mouse expression of complement receptor-3 (CR3, α M β 2 integrin). Chilled platelets injected into mice lacking CR3 showed normal hemostatic function. Removal of the extracellular domain of GPIBα, part of the receptor for von Willebrandt factor (vWf) that also binds CR3, abolished rapid clearance and reduced phagocytosis of chilled human platelets. Chilling caused clustering of immunogold-labeled GPIBα but did not affect binding of activated vWf. Platelet counts in mice with CR3 receptors dropped when they were exposed to cold temperatures, whereas platelet counts in mice lacking CR3 did not. Mild chilling primes platelets for activation, which the authors previously proposed limits bleeding at exposed surfaces; in this paradigm, rapid clearance prevents thrombosis in critical core regions by these primed platelets. K. M. Hoffmeister, T. M. Felbinger, H. Falet, C. V. Denis, W. Bergmeier, T. N. Mayadas, U. H. von Andrian, D. D. Wagner, T. P. Stossel, J. H. Hartwig, The clearance mechanism of chilled blood platelets. Cell 112 , 87-97 (2003). [Online Journal]