Growth and Stability of Thrombi in Flowing Citrated Blood: Assessment of Platelet-Surface Interactions with Computer-Assisted Morphometry

Summary

The differential quantitation of platelet deposition in perfusion studies is a major problem. We report on methods to prepare semithin sections of platelet deposits on collagen coated on glass and plastic cover slips, to study growth and stability of thrombi in three dimensions, and the development of a computer-assisted differential quantitation of platelet-collagen interactions. The interactions were quantified as percentage of the surface covered with platelets (platelet adhesion), thrombus height, thrombus density and thrombus area per unit sectional length, respectively.

Cover slips coated with fibrillar equine collagen in parallelplate perfusion chambers were exposed to flowing citrated blood at shear rates ranging from 200 to 2,600 s⁻¹. Thrombi, partially enmeshed in the collagen meshwork, prevailed on the surface at all shear rates. Maximal platelet adhesion and thrombus density were seen at >5 μg/cm² collagen, while thrombus area and height were maximal at >10 μg/cm². The volume of the thrombi appeared correlated to the number of deposited platelets (r = 0.92). En face preparations showed deposits of platelet islands which grew in diameter with time, particularly in the direction of the blood flow, becoming progressively confluent. Sections cut parallel to the direction of the blood stream indicated that this growth pattern was at least partially caused by thrombi bent in the direction of the blood flow. This view is consistent with data from corresponding sections cut perpendicular to the direction of the blood flow showing that the initial thrombus growth at 2 min is isotropic, while anisotropic growth, characterized with decreased growth in height, is observed at 5 and 10 min.

Our three-dimensional analysis suggests that the growth occurs mainly in height, and that blood shear forces may bend the thrombi toward the surface resulting in platelet thrombi preferentially elongated in the direction of the blood flow.

Key words

Perfusion chamber - Shear rate - Thrombus growth - Thrombus stability - Computer-assisted morphometry

• References

• 1 Spaet TH, Lejnieks I. A technique for estimation of platelet-collagen adhesion. Proc Soc Exp Med 1969; 132: 1038-1041
• 2 Born G VR. Observations on the change in shape of blood platelets brought about by adenosine diphosphate. J Physiol 1970; 209: 487-511
• 3 Cazenave JP, Packham MA, Mustard JF. Adherence of platelets to a collagen coated surface: development of a quantitative method. J Lab Clin Med 1973; 82: 978-990
• 4 Tschopp TB. Aspirin inhibits platelet aggregation on, but not adhesion to, collagen fibrils: an assessment of platelet adhesion and deposited platelet mass by morphometry and 51Cr-labeling. Thromb Res 1977; 11: 619-632
• 5 Muggli R, Baumgartner HR, Tschopp TB, Keller H. Automated microdensitometry and protein assays as a measure for platelet adhesion and aggregation on collagen coated slides under controlled flow conditions. J Lab Clin Med 1980; 95: 195-207
• 6 Sakariassen KS, Aarts P A MM, de Groot PhG, Houdijk W PM, Sixma JJ. A perfusion chamber developed to investigate platelet interaction in flowing blood with human vessel wall cells, their extracellular matrix, and purified components. J Lab Clin Med 1983; 102: 522-535
• 7 Adams GA, Feuerstein IA. Platelet accumulation on collagen: drugs which inhibit arachidonic acid metabolism and affect intracellular cyclic AMP levels. Thromb Haemostas 1984; 52: 45-49
• 8 Houdijk W PM, Sakariassen P KS, Nievelstein F EM, Sixma JJ. Role of factor VUI-von Willebrand factor and fibronectin in the interaction of platelets in flowing blood with monomeric and fibrillar human collagen types I and III. J Clin Invest 1985; 75: 531-540
• 9 Legrand YJ, Fauvel F, Kartalis G, Wautier JL, Caen JP. Specific and quantitative method for estimation of platelet adhesion to fibrillar collagen. J Lab Clin Med 1979; 94: 438-446
• 10 Baumgartner HR, Muggli R. Adhesion and aggregation: morphologically demonstration and quantitation in vivo and in vitro. In: Platelets in Biology and Pathology Gordon JL. (ed) Elsevier/North-Holland Biomedical Press; Amsterdam: 1976: 23
• 11 Turitto VT, Muggli R, Baumgartner HR. Platelet adhesion and thrombus formation on subendothelium, epon, gelatin and collagen under arterial flow conditions. Am Soc Artif Intern Organs 1979; 2: 28-34
• 12 Baumgartner HR. Platelet interaction with collagen fibrils in flowing blood. I. Reaction of human platelets with a-chymotrypsin-digested subendothelium. Thromb Haemostas 1977; 37: 1-14
• 13 Turitto VT, Baumgartner HR. Platelet-surface interaction. In: Hemostasis and Thrombosis, Basic Principles and Clinical Practice Colman RW, Hirsh J, Marder V, Salzman EW. (eds) J B Lippincott Company; Philadelphia PA: 1982: 364
• 14 Sakariassen KS, Bolhuis PA, Sixma JJ. Human blood platelet adhesion to artery subendothelium is mediated by factor VIII-von Willebrand factor bound to the subendothelium. Nature 1979; 279: 636-638
• 15 Sakariassen KS, Fressinaud E, Girma JP, Baumgartner HR, Meyer D. Mediation of platelet adhesion to fibrillar collagen in flowing blood by a proteolytic fragment of human von Willebrand factor. Blood 1986; 67: 1515-1518
• 16 Sakariassen KS, Bolhuis PA, Sixma JJ. Platelet adherence to subendothelium of human arteries in pulsatile and steady flow. Thromb Res 1980; 19: 547-559
• 17 Baumgartner HR. The role of blood flow in platelet adhesion, fibrin deposition and formation of mural thrombi. Micro vase Res 1973; 5: 167-179
• 18 Sakariassen KS, Muggli R, Baumgartner HR. Measurements of platelet interaction with components of the vessel wall in flowing blood. In: Methods in Enzymology Hawiger JJ. (ed) Academic Press Inc; New York: 1988. 169 137
• 19 Baumgartner HR, Turitto VT, Weiss HJ. Effect of shear rate on platelet interaction with subendothelium in citrated and native blood. II. Relationships among platelet adhesion, thrombus dimensions, and fibrin formation. J Lab Clin Med 1980; 95: 208-221
• 20 Weibel ER. Volume density measurement. In: Academic Press Inc; London: 1979: 26 Weibel ER. (ed) Stereological Methods, Volume 1
• 21 Baumgartner HR, Sakariassen KS. Factors controlling thrombus formation on arterial lesions. Ann New York Acad Sci, New York 1985; 454: 162-177
• 22 Sakariassen KS, Baumgartner HR. Axial dependence of platelet deposition on collagen in flowing blood: Upstream thrombus growth impairs downstream platelet adhesion. Arteriosclerosis 1989. (in press)
• 23 Bolhuis PA, Sakariassen KS, Sander HJ, Bouma BN, Sixma JJ. Binding of factor VIII-von Willebrand factor to human subendothelium precedes increased platelet adhesion and enhances platelet spreading. J Lab Clin Med 1981; 97: 568-576
• 24 Adams GA, Brown SJ, McIntyre LV, Eskin SG, Martin RR. Kinetics of platelet adhesion and thrombus growth. Blood 1983; 62: 69-74
• 25 Weiss HJ, Turitto VT, Vicic WJ, Baumgartner HR. Fibrin formation, fibrinopeptide A release, and platelet thrombus dimensions on subendothelium exposed to flowing native blood: Greater in factor XII and XI than in factor VIII and IX deficiency. Blood 1984; 63: 1004-1014