Absence of Fibrinogen in Afibrinogenemia Results in Large but Loosely Packed Thrombi under Flow Conditions

Summary

We studied the role of fibrinogen in platelet thrombus formation under flow on adhesive proteins using afibrinogenemic blood (LMWH anticoagulated) in a perfusion system. Perfusions with afibrinogenemic blood showed strong increased surface coverage and thrombus volume that normalized upon addition of fibrinogen. Similar studies using citrate anticoagulated blood showed that this was due to fibrinogen and not fibrin. Morphological analysis showed that afibrinogenemic thrombi were loosely packed and consisted mainly of dendritic platelets that contacted one another through filopodia. However, in the presence of fibrinogen, platelets formed lamellipodia and spread out on top of one another. Studies with radiolabeled platelets showed similar numbers of platelets in both conditions demonstrating that the difference is one of packing and the larger size is due to absence of lamellipodia formation and spreading. The found increased thrombus size and loosely packed platelets might help to understand thrombotic complications sometimes seen in afibrinogenemia patients.

Keywords

Afibrinogenemia - thrombus - flow - fibrinogen - platelet

• References

• 1 Soria J, Soria C, Borg JY, Mirshahi M, Piguet H, Tron P, Fessard C, Caen JP. Platelet aggregation occurs in congenital afibrinogenaemia despite the absence of fibrinogen or its fragments in plasma and platelets, as demonstrated by immunoenzymology. Br J Haematol 1985; 60: 503-14.
• 2 Al-Mondhiry H, Ehmann WC. Congenital afibrinogenemia. Am J Hematol 1994; 46: 343-7.
• 3 Tollefsen DM, Majerus PW. Inhibition of human platelet aggregation by monovalent antifibrinogen antibody fragments. J Clin Invest 1975; 55: 1259-68.
• 4 Weiss HJ, Rogers J. Fibrinogen and platelets in the primary arrest of bleeding. Studies in two patients with congenital afibrinogenemia. N Engl J Med 1971; 285: 369-74.
• 5 Gralnick HR, Williams SB, Coller BS. Fibrinogen competes with von Willebrand factor for binding to the glycoprotein IIb/IIIa complex when platelets are stimulated with thrombin. Blood 1984; 64: 797-800.
• 6 Piétu G, Cherel G, Marguerie G, Meyer D. Inhibition of von Willebrand factor-platelet interaction by fibrinogen. Nature 1984; 308: 648-49.
• 7 Turitto VT, Weiss HJ, Baumgartner HR. Platelet interaction with rabbit subendothelium in von Willebrand’s disease: altered thrombus formation distinct from defective platelet adhesion. J Clin Invest 1984; 74: 1730-41.
• 8 Weiss HJ, Hawiger J, Ruggeri ZM, Turitto VT, Thiagarajan P, Hoffmann T. Fibrinogen-independent platelet adhesion and thrombus formation on subendothelium mediated by glycoprotein IIb-IIIa complex at high shear rate. J Clin Invest 1989; 83: 288-97.
• 9 Goto S, Ikeda Y, Saldivar E, Ruggeri ZM. Distinct mechanisms of platelet aggregation as a consequence of different shearing flow conditions. J Clin Invest 1998; 101: 479-86.
• 10 Caen J, Faur Y, Inceman S. et al. Necrose ischémique bilatérale dans un cas de grande hypofibrinogénémie congénitale. Nouv Rev Fr Hématol 1964; 4: 321-4.
• 11 Marchal G, Duhamel G, Samama M, Flandrin G. Thrombose massive des vaisseaux d’un membre au cours d‘une hypofibrinémie congénitale. Hémostase 1964; 4: 81-90.
• 12 Nilsson IM, Nilehn JE, Cronberg S, Norden G. Hypofibrinogenaemia and massive thrombosis. Acta Med Scand 1966; 180: 65-76.
• 13 Molho-Sabatier P, Soria C, Legrand C. et al. Fibrinogen independent platelet aggregation responsible for arterial thrombosis in an afibrinogenemic patient [abstract]. Thromb Haemost 1991; 65: 850.
• 14 Lak M, Keihani M, Elahi F, Peyvandi F, Mannucci PM. Bleeding and thrombosis in 55 patients with inherited afibrinogenaemia. Br J Haematol 1999; 107: 204-6.
• 15 Sodetz JM, Pizzo SV, McKee PA. Relationship of sialic acid to function and in vivo survival of human factor VIII/von Willebrand factor protein. J Biol Chem 1977; 252: 5538-46.
• 16 Wu YP, Vink T, Schiphorst M, IJsseldijk MJW, van Zanten GH, de Groot PG, Sixma JJ. Platelet thrombus formation on collagen at high shear rate is mediated by von Willebrand factor (vWF)-GPIb interaction and inhibited by vWF- α_IIbβ₃ interaction. Arterioscler Thromb Vasc Biol 2000; 1661-7.
• 17 Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest 1973; 52: 2745-56.
• 18 Willems C, Astaldi GC, de Groot PG, Janssen MC, Gonsalvez MD, Zeijle-maker WP, Van Mourik JA, Van Aken WG. Media conditioned by cultured human vascular endothelial cells inhibit the growth of vascular smooth muscle cells. Exp Cell Res 1982; 139: 191-7.
• 19 Zwaginga JJ, Sixma JJ, de Groot PG. Activation of endothelial cells induces platelet thrombus formation on their matrix. Studies of new in vitro thrombosis model with low molecular weight heparin as anticoagulant. Arteriosclerosis 1990; 10: 49-61.
• 20 Sixma JJ, de Groot PG, van Zanten H, IJsseldijk M. A new perfusion chamber to detect platelet adhesion using a small volume of blood. Thromb Res 1998; 92: S43-S46.
• 21 Sakariassen KS, Aarts PA, de Groot PG, Houdijk WP, 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: 522-35.
• 22 Dise CA, Burch JW, Goodman DB. Direct interaction of mepacrine with erythrocyte and platelet membrane phospholipid. J Biol Chem 1982; 257: 4701-4.
• 23 Savage B, Saldivar E, Ruggeri ZM. Initiation of platelet adhesion by arrest onto fibrinogen or translocation on von Willebrand factor. Cell 1996; 84: 289-97.
• 24 Tsuji S, Sugimoto M, Miyata S, Kuwahara M, Kinoshita S, Yoshioka A. Real-time analysis of mural thrombus formation in various platelet aggregation disorders: distinct shear-dependent roles of platelet receptors and adhesive proteins under flow. Blood 1999; 94: 968-75.
• 25 Ruggeri ZM, Dent JA, Saldivar E. Contribution of distinct adhesive interactions to platelet aggregation in flowing blood. Blood 1999; 94: 172-8.
• 26 Baumgartner HR. The role of blood flow in platelet adhesion, fibrin deposition, and formation of mural thrombi. Microvascular Research 1973; 5: 167-79.
• 27 Remijn JA, Sixma JJ, de Groot PG. Fibrin formation under conditions of flow results in fibrin fibers carried along by flow and deposited downstream to preadsorbed fibrinogen [abstract]. Thromb Haemost 1999; 82: 41.
• 28 Chafa O, Chellali T, Sternberg C, Reghis A, Hamladji RM, Fischer AM. Severe hypofibrinogenemia associated with bilateral ischemic necrosis of toes and fingers. Blood Coagul Fibrinolysis 1995; 6: 549-52.
• 29 Ni H, Denis CV, Subbarao S, Degen JL, Sato TN, Hynes RO, Wagner DD. Persistence of platelet thrombus formation in arterioles of mice lacking both von Willebrand factor and fibrinogen. J Clin Invest 2000; 106: 385-92.