The platelet laminin receptor : discovery of a 67kDA receptor for laminin on the membranes of human platelets : characterisation and isolation

Doctoral Thesis


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University of Cape Town

Previous work on the binding of resting platelets to the basement membrane glycoprotein, laminin, has identified the Ic/IIa integrin c01aplex (CD49f/CD29), also known as VLA-6, as the receptor. There exists however, another protein with a molecular weight of 67kDa, that mediates this function on other cells. It is abundantly expressed on the membranes of breast cancer cells, where it plays a key role in both the localisation at, and penetration of vascular beds, by metastases. The objectives of this study were: * The development of a micro-titre assay similar to those used in previous studies, standardised and calibrated to characterize the adhesion of unstimulated normal human platelets to laminin-coated surfaces. * To determine the effect on adhesion of platelet activation, enzymatic surface-glycoprotein removal, antibodies to specific receptors and interaction with other adhesive proteins known to bind to platelet membranes. * To establish the in vivo relevance of the experimental findings, by the assay of adhesion of glycoprotein IIb/IIIa-deficient platelets of two patients with Glanzmann's Thrombasthenia. These studies serve d to distinguish specific binding sites for laminin from the known surface receptors of platelets. The methodology used to isolate laminin receptors from the membranes of breast carcinoma cells was then applied to platelet concentrates. Membranes were obtained by centrifuging the ultrasonic lysate of a unit of platelets. These were solubilized and passed over a laminin-Sepharose column. The bound components were eluted and identified by means of SDS-gel electrophoresis, after which a concentrate was tested for laminin binding by means of dot-blot methodology. The principle contribution of this work is the finding of a 67kDa receptor for laminin on the surface membranes of platelets. The combination of the various approaches applied to characterise the adhesion of platelets to laminin, show that this is a specific, Mg²⁺-dependent process, inhibited by Ca²⁺ and not enhanced by platelet activation. Adhesion was decreased by proteolysis with trypsin and chymotrypsin, showing that the adhesion is mediated by a surface glycoprotein. Proteolysis with the Serratia marcescens metalloprotease, which cleaves off glycoprotein lb, did not affect adhesion, proving that this well-known receptor for platelet adhesion is not involved in the adhesion. The receptors GPIV and glycocalicin were also excluded, as the presence of antibodies to these receptors had no effect. Prior incubation with fibrinogen or von Willebrand factor, which binds to specific receptors on the platelet membrane, inhibited adhesion, most likely due to spatial interference with the receptor site for laminin. The presence of the tetrapeptide recognised by the membrane receptors for many adhesive proteins, RGDS, at concentrations of up to 1mM, had no effect. The platelets of the two subjects with Glanzmann's Thrombasthenia adhered normally, definitively ruling out the involvement of GPIIb/IIIa, which is absent from these platelets. The isolation process recovered a membrane component from the laminin-Sepharose column with an elution pattern identical to that for the well characterised 67kDa receptor for laminin on the surface of breast carcinoma cells. They have the same molecular weights in both the reduced (67kDa) and non-reduced (53kDa) states. Blot identification demonstrated laminin binding by the eluate. In the last part of the work, collaborative studies using more sophisticated methodology have confirmed that platelet receptors for laminin play a role in their adhesion to living tissue. Anti-laminin Fab antibodies significantly decreased the adhesion when whole blood was perfused over isolated rabbit aortic segments. That these receptors are identical to the 67kDa receptor of breast carcinoma cells was shown by the specific, high affinity binding of antibodies directed at the carcinoma receptors to the surface of platelets when examined by flow cytometry. In addition, they inhibit platelet adhesion by 50-60% in the micro-titre assay. It is proposed that both the VLA-6 and the 67kDa receptors are required for platelet adhesion to laminin, possibly as a two-stage process, similar to the systems for adhesion to von Willebrand factor, where binding is initially to GPIb, followed by binding to GPIIb/IIIa. The possible relevance of this receptor in the pathophysiology of the metastatic process is discussed.