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Thrombospondin-1 mediates platelet adhesion at high shear via glycoprotein Ib (GPIb): an alternative/backup mechanism to von Willebrand factor¹

KERSTIN JURK, KENNETH J. CLEMETSON^*, PHILIP G. DE GROOT, MARTIN F. BRODDE, MICHAEL STEINER, NAPHTALI SAVION, DAVID VARON^||, JAN J. SIXMA, HUGO VAN AKEN and BEATE E. KEHREL²

Department of Anaesthesiology and Intensive Care, Experimental and Clinical Haemostasis, University-Hospital Münster, Germany;
^* Theodor Kocher Institute, University of Berne, Switzerland;
Department of Haematology, University Medical Centre Utrecht, Utrecht, Netherlands;
Institute of Clinical Chemistry, University of Rostock, Germany;
Goldschleger Eye Research Institute, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel; and
^|| Coagulation Unit, Department of Hematology, Hadassah Hebrew University Medical Center, Jerusalem, Israel

²Correspondence: Department of Anaesthesiology and Intensive Care, Experimental and Clinical Haemostasis, University-Hospital Münster, Mendelstr. 11, 48149 Münster, Germany. E-mail: kehrel{at}uni-muenster.de

SPECIFIC AIMS

Acute thrombotic arterial occlusion is the leading cause of morbidity and mortality in the Western world and the von Willebrand factor (VWF) is thought to be the only indispensable adhesive substrate to promote thrombus formation under conditions of high shear as found in stenosed coronary arteries. Since platelets also adhere in a VWF-deficient system, we looked for an alternative substrate to allow thrombus formation under high shear conditions.

PRINCIPAL FINDINGS

1. Thrombospondin-1 mediates extremely firm platelet adhesion at high shear
Thrombospondin-1 (TSP-1) supported platelet adhesion in LMWH-anti-coagulated whole blood in both devices used: the parallel plate perfusion chamber (PPC) and the cone and plate(let) analyzer (CPA). When shear rate was increased from static to 1600–1800 s^–1, platelet adhesion to TSP-1 increased significantly. No adhesion was observed when Ca²⁺ was removed. High shear platelet adhesion to TSP-1 is extremely firm, with no significant detachment of platelets at shear rates of up to 4000 s^–1. Second perfusions in the absence of Ca²⁺ immediately resulted in complete platelet detachment.

2. Platelet adhesion to TSP-1 at high shear is independent of VWF
TSP-1 binds to many adhesive proteins, including VWF, the known partner for platelet high shear adhesion. We therefore examined whether platelet adhesion to TSP-1 at the high shear rate of 1700 s^–1 is mediated indirectly via a VWF bridge between TSP-1 and the platelet surface. We assessed platelet adhesion to TSP-1 using blood from a patient with von Willebrand’s disease (VWd) type 3, lacking detectable VWF in both plasma and platelets. In the absence of VWF, platelets adhered to TSP-1 perfectly normally.

3. Nature of the "high shear adhesion TSP-1 receptor"
Several platelet membrane proteins and proteoglycans have been proposed as TSP-1 receptors, including the integrins ₂ß₁ (GPIa/IIa), _IIbß₃ (GPIIb/IIIa), and _vß₃; CD36; the integrin-associated protein (IAP, CD47); and heparan sulfate. Neither RGDS peptides, other GPIIb/IIIa and _vß₃ antagonists (antibodies 7E3, P2), nor replacement of normal platelets with thrombasthenic platelets lacking GPIIb/IIIa and _vß₃ affected the adhesion of platelets to TSP-1 at the high shear rate of 1600 s^–1 or 1700 s^–1. Platelet surface coverage on TSP-1 at 1700 s^–1 did not depend on ₂ß₁ integrin. P1E6 antibody, which blocks platelet adhesion to type I collagen at the concentration used, had no inhibitory effect, and ₂ß₁-deficient platelets adhered normally to TSP-1. Platelet high shear adhesion to TSP-1 did not differ between heparin- and hirudin-anticoagulated blood, and soluble heparan sulfate did not influence the adhesion rate. Neither the functional blocking antibody CIKM1 against CD47 nor preincubation of the TSP-1 surface with anti-TSP-1 antibody C6.7 recognizing the CD47 binding site in TSP-1 influenced platelet adhesion to TSP-1 at 1600 s^–1. Two of the monoclonal antibodies against CD36, AmAK5 and OKM5, partly inhibited platelet adhesion to TSP-1. Increasing the inhibitory antibody concentration above 10 µg/mL did not inhibit further. Antibodies RAG-35 and AVW3 against the GPIb binding site of VWF, which completely block platelet adhesion to VWF at this shear rate at the concentration used, had only a partial inhibitory effect in experiments when VWF was present. Higher concentrations of these antibodies did not inhibit further.

4. GPIb is the direct platelet counter receptor for TSP-1 high shear interaction
GPIb has specific properties especially suitable for binding ligands to withstand high shear. We therefore tested whether GPIb directly mediates adhesion to immobilized TSP-1 under these shear conditions. In both adhesion devices, several antibodies against GPIb at 10 µg/mL inhibited almost completely (Fig. 1 A, B–E). We confirmed the specific role of GPIb as high shear counter receptor for TSP-1 using glycocalicin, the soluble extracellular domain of GPIb, which inhibited platelet adhesion to TSP-1 up to 80% (Fig. 1A, F ). In an ELISA system, TSP-1 bound to immobilized glycocalicin specifically in a concentration-dependent manner.

View larger version (77K): [in this window] [in a new window]   Figure 1. GPIb as high shear counter receptor on platelets for TSP-1. A) LMWH blood was incubated with several antibodies against GPIb (clone 2128, G.19H10, SZ2, AP1, blocking GPIb–VWF interaction) before applying shear stress in CPA (1700 s–1). Data are from 3 independent experiments, mean ± SD (*P<0.05, **P<0.001 vs. control). B–F) Inhibition of platelet adhesion to TSP-1 (1700 s–1, CPA) by functional blocking mAbs against GPIb and by glycocalicin. Adherent platelets on TSP-1 matrix were stained and evaluated by inverted light microscopy.

CONCLUSIONS AND SIGNIFICANCE

VWF is thought to be the unique, indispensable adhesive substrate for thrombus formation in high shear environments like stenosed coronary arteries, but recently it was shown that the matrix of HMEC-1 cells supports thrombus formation at high shear in the absence of VWF. It has been shown in vivo with mice lacking both VWF and fibrinogen that VWF is inessential for platelet adhesion to subendothelium at arterial shear (>1300 s^–1). Fibronectin from the -granules is believed to be the ligand. However, platelet adhesion to fibronectin has an optimum at 300 s^–1 and at arteriole shear rates platelets do not adhere to fibronectin. We identified TSP-1 as alternative substrate to promote thrombus formation under high shear conditions up to a shear rate of 1900 s^–1. Platelets adhere to immobilized TSP-1 in a shear-dependent manner with an optimum shear as found in stenosed arteries. This adhesion is extremely firm, with no detachment of platelets up to a shear rate of 4000 s^–1 or 118 dynes/cm². Regarding platelet adhesion at pathophysiological shear rates higher than 2000 s^–1, however, there is no doubt that VWF is the predominantly adhesive substrate for thrombus formation. It was reported that platelets adhering to fibronectin and laminin were most easily detached and that detachment tended to be linear with respect to shear stress. Thus, TSP-1 is at least one of the components in the subendothelium that strengthens its shear resistance. In contrast, platelet adhesion to fibronectin platelet-mediated adhesion to TSP-1 does not depend on VWF as shown using plasma and platelets lacking VWF. TSP-1 is a potent inhibitor of angiogenesis acting directly on endothelial cells via CD36. Although it has been observed that CD36 blocking antibody OKM5 had no effect on static platelet adhesion to TSP-1, we observed partial inhibition under flow conditions. The difference between static and flow conditions might be explained by the fact that the platelets must be preactivated to allow CD36 binding to TSP-1. This suggests that CD36 is not a primary TSP-1 high shear adhesion receptor but is involved in interaction and signal transduction via TSP-1 in preactivated platelets.

Since platelet adhesion to TSP-1 is extremely firm, we tested GPIb as primary high shear adhesion receptor for TSP-1 and identified GPIb as the platelet counter receptor. Other proteins or proteoglycans, heparan sulfate, integrin _IIbß₃, _vß₃, ₂ß₁, and integrin-associated protein (IAP, CD47) known to bind TSP-1 were excluded as platelet high shear adhesion receptors. Our results might suggest similar binding sites on TSP-1 and VWF for interaction with GPIb. There is 27.3% identity in 33 aa between the VWF A1 domain and TSP-1 amino terminus amino acids 136-170 (PIR pairwise alignment, Smith-Waterman score: 49) and a 24% identity in a 50 aa-overlap (Smith-Waterman score: 41) between the VWF A1 domain and the type I repeats of TSP-1.

In conclusion, TSP-1 might be important as an adhesive substrate in microvessels in vivo, where neither VWF nor fibrillar collagens that strongly bind plasma or platelet VWF are present. We strongly believe that our findings contribute fundamentally to understanding of the molecular basis of acute thrombotic arterial occlusion (Fig. 2 ). These findings suggest that the thrombospondin-1/GPIb axis is an interesting therapeutic target. The newly discovered GPIb-TSP-1 adhesion axis under arterial shear conditions might be important not only during thrombus formation, but for pathological processes including arteriosclerosis, inflammation, and tumor metastasis, where other cells bind to the endothelium.

View larger version (41K): [in this window] [in a new window]   Figure 2. Schematic representation of the mechanism of platelet adhesion and aggregation in flowing blood under high shear conditions. A) In the classical view, VWF mediates platelet adhesion and aggregation in flowing blood under high shear conditions. B) TSP-1 acts as an alternative/backup substrate under high shear conditions and uses the same direct platelet receptor as VWF, glycoprotein Ib (GPIb).

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0830fje; doi: 10.1096/fj.02-0830fje

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