The Basement Membrane Underlying the Vascular Endothelium Is Not Thrombogenic: In Vivo and In Vitro Studies with Rabbit and Human Tissue

Summary

Studies examining the interaction of platelets with exposed subendothelium in vivo have reported conflicting results, lo examine possible explanations for the apparently discrepant findings, we measured the platelet reactivity of subendothelium prepared by a number of methods both in vivo and in vitro. In addition, we examined the possibility that 13-hydroxyoc-tadecadinoic acid (13-HODE), an endothelial cell-derived chemorepellant, modulates the reactivity of the subendothelium to platelets. In vivo, the subendothelium of segments of rabbit carotid arteries was exposed by removing the endothelial cells by air perfusion or by balloon catheter stripping. Platelet accumulation onto the dc-cndothelialized segments was assessed by ³H-radioaclivily uptake, using ³H-adenine-labelled platelets, and by scanning electron microscopy. In vitro, ³H-adenine-labelled platelet adhesion was measured onto plain plastic discs and onto plastic discs coated with the following purified basement membrane components: collagens type I, III, IV, V, laminin, or fibronectin. In addition, ³H-adenine-labelled platelet adhesion was measured onto plastic discs coveredwith human endothelial cells or onto the basement membrane underlying the endothelial cells.

In vivo, there was marked ³H-platelet accumulation onto the ballon catheter carotid arteries one hour after injury. In contrast, there was no platelet accumulation onto the subendothelium of carotid arteries de-endothelialized by air perfusion. These differences were confirmed by scanning electron microscopy. Transmission electron microscopic examination demonstrated that the extracellular matrix was intact following the air perfusion injury whereas the majority of it was removed by the balloon catheter injury.

In vitro, the accumulation of ³H-platelets onto plain discs and onto discs coated with any of the four collagens, fibronectin or laminin was significantly greater than the adhesion of ³H-platelets onto intact endothelial cells or the basement membrane prepared by cellulose acetate stripping. In contrast, ³H-platelet adhesion onto the basement membrane prepared by ammonium hydroxide treatment was significantly increased. An HPLC analysis of methanol extracts obtained from the two basement membranes and the cultured endothelial in vitro demonstrated that there was significant amounts of 13-HODE present in the endothelial cells and in the basement membrane prepared by the mechanical stripping, but there was no detectable 13-HODE in the basement membrane prepared by ammonium hydroxide treatment.

We conclude that platelets do not adhere to subendothelium immediately beneath the endothelium and that this thromboresistance is contributed to by 13-HODE. We also suggest that the observed thrombogeneicity of subendothelium following balloon-induced injury is due to the mechanical removal of sub-endothelial structures including 13-HODE, exposing deeper more thrombogenic vascular wall structures.

Key words

Basement membrane - 13-hydroxy-octadecadienoic acid (13-HODE) - Vascular thromboresistance

• References

• 1 Ross R, Gomset JA. The pathogenesis of atherosclerosis. N Engl J Med 1976; 295: 369-377 420-25
• 2 Stemerman MB. Relationship of thrombosis to atherosclerosis. In: Thrombosis and Haemostasis, Basic Principles and Usual Practice. Coleman RW, Hirsh J, Marder V, Salzman EW. (eds.) Lippincott J B; Philadelphia: 1982. pp 781-788
• 3 Tschopp TB, Baumgartner HR, Silberbauer K, Sinzinger H. Platelet adhesion and platelet thrombus formation on subendothelium of human arteries and veins exposed to flowing blood in vitro: a comparison with rabbit aorta. Haemostas 1979; 8: 19-29
• 4 Buchanan MR, Richardson M, Vallee E. Basement membrane underlying the vascular endothelium is not thrombogenic. Clin Invest Med 1985; 8 (Suppl B) 27A
• 5 Tissinier A, Vallee E, Bernat A, Maffrand J-P. Kinetics of platelet adhesion to rat carotid artery following selective deendothelialisation by air injury. Biol Cell 1984; 51: 3A
• 6 Madri JA, Drey B, Pitlick FA, Furthmayr H. The collagenous components of the subendothelium: correlation of structure and function. Lab Invest 1980; 43: 303-315
• 7 Furthmayr H, Roll FJ, Madri JA, Foellmer HG. Composition of basement membranes as viewed with the electron microscope. In: New Trends in Basement Membrane Research. Raven Press; New York: 1982. pp 31-48
• 8 Stemerman MB, Baumgartner HR, Spaet TH. The subendothelial microfibril and platelet adhesion. Lab Invest 1971; 24: 179-186
• 9 Trelstad RL. Special state of the fibril end: site of growth, point of cell surface attachment, and possible site for platelet interaction. In Collagen-Platelet Interactions. Gastpar H. (ed.) Schattauer Verlag; New York: 1978. p 153
• 10 Mason RG, Mohammad SF, Chuan HYK, Richardson PD. The adhesion of platelets to subendothelium, collagen and artificial surfaces. Semin Thrombos Ilaemostas 1976; 3: 98-116
• 11 Buchanan MR, Haas TA, Lagardc M, Guichardant M. 13-hydroxyoctadecadienoic acid is the vessel wall chemorepellent factor, LOX. J Biol Chem 1985; 260: 16,056-9
• 12 Madri JA, Furthmayr H. Isolation and tissue localization of type AB? collagen from normal lung parenchyma. Amer J Pathol 1979; 94: 323-331
• 13 Madri JA, Roll FJ, Furthmayr H, Foidart J-M. The ultrastructural localization of fibronectin and laminin in the basement membrane of the murine kidney. J Cell Biol 1980; 86: 682-687
• 14 Foellmer HG, Madri JA, Furthmayr H. Monoclonal antibodies to type IV collagen: Probes for the study of structure and function of basement membranes. Lab Invest 1983; 48: 639-649
• 15 Enguall E, Ruoslahti E. Binding of soluble form of fibroblast surface protein, fibronectin, to collagen. Int J Cancer 1977; 20: 1-5
• 16 Buchanan MR, Butt RW, Magas Z, Van Ryn J, Hirsh J, Nazir DJ. Endothelial cells produce a lipoxygenase derived chemorepellent which influences platelet/endothelial cell interactions. Effect of aspirin and salicylate. Thrombos Haemostas 1985; 53: 306-311
• 17 Fishman JA, Ryan GB, Karnovsky MJ. Endothelial regeneration in the rat carotid artery and the significance of endothelial denudation in the pathogenesis of myointimal thickening. Lab Invest 1975; 32: 339-351
• 18 Buchanan MR, Hirsh J. The effect of aspirin and salicylate on platelet vessel wall interaction in rabbits. Atherosclerosis 1984; 4: 403-406
• 19 Spurr AR. Low-viscosity embedding media. J Ultrastruct Res 19,699 26: 31-43
• 20 Form DM, Pratt BM, Madri JA. Endothelial cell proliferation during angiogenesis: In vitro modulation by basement membrane components. Lab Invest 1986; 55: 521-530
• 21 Buchanan MR, Dejana E, Gent M, Mustard JF, Hirsh J. Enhanced platelet accumulation onto carotid arteries in rabbits following aspirin treatment. J Clin Invest 1981; 67: 503-508
• 22 Borgeat P, de Laclos BF, Rabinovitch H, Picard S, Braquret P, Hebert J, Lavioette M. Eosinophil-rich human polymorphonuclear leukocyte preparations characteristically release leukotriene C₄ on ionophore A23187 challenge. J Allergy Clin Immunol 1984; 74: 310-315
• 23 Hambcrg M, Samuclsson B. On the specificity of the oxygenation of unsaturated fatty acids catalyzed by soybean lipoxidase. J Biol Chem 1967; 242: 5329-5335
• 24 Wang CL, Miyata T, Weksler B. Collagen induced platelet aggregation and release. II. Arterial size and structural requirements of collagen. Biochim Biophys Acta 1978; 544: 568-577
• 25 Groves HM, Kinlough-Rathbone RL, Richardson M, Moore S, Mustard JF. Platelet interaction with damaged rabbit aorta. Lab Invest 1979; 40: 194-200