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DOI: 10.1055/s-0038-1653771
Antithrombotic Effect of a Recombinant von Willebrand Factor, VCL, on Nitrogen Laser-Induced Thrombus Formation in Guinea Pig Mesenteric Arteries
Publikationsverlauf
Received 13. April 1994
Accepted after revision 19. Oktober 1994
Publikationsdatum:
09. Juli 2018 (online)
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Summary
To assess the antithrombotic effectiveness of blocking the platelet glycoprotein (GP) Ib/IX receptor for von Willebrand factor (vWF), the antiaggregating and antithrombotic effects were studied in guinea pigs using a recombinant fragment of vWF, Leu 504-Lys 728 with a single intrachain disulfide bond linking residues Cys 509-Cys 695. The inhibitory effect of this peptide, named VCL, was tested in vitro on ristocetin- and botrocetin-induced platelet aggregation and compared to the ADP-induced platelet aggregation. In vivo, the antithrombotic effect of VCL was tested in a model of laser-injured mesentery small arteries and correlated to the ex vivo ristocetin-induced platelet aggregation. In this model of laser-induced thrombus formation, five mesenteric arteries were studied in each animal, and the number of recurrent thrombi during 15 min, the time to visualization and time to formation of first thrombus were recorded.
In vitro, VCL totally abolished ristocetin- and botrocetin-induced platelet aggregation, but had no effect on ADP-induced platelet aggregation. Ex vivo, VCL (0.5 to 2 mg/kg) administered as a bolus i. v. injection inhibits ristocetin-induced platelet aggregation with a duration of action exceeding 1 h. The maximum inhibition was observed 5 min after injection of VCL and was dose related. The same doses of VCL had no significant effect on platelet count and bleeding time. In vivo, VCL (0.5 to 2 mg/kg) had no effect on the appearance of the thrombi formed but produced dose-dependent inhibition of the mean number of recurrent thrombi (the maximal effect was obtained at 5 min following i. v. injection of the highest dose: 0.8 ± 0.2 thrombi versus 4 ± 0.4 thrombi in controls). The three doses of VCL increased the time in which the first thrombus in a concentration-dependent manner was formed. However, the time to visualize the first thrombus was only prolonged in the higher dose-treated group.
These in-vivo studies confirm that VCL induces immediate, potent, and transient antithrombotic effects. Most importantly, this inhibition was achieved without inducing thrombocytopenia nor prolongation of the bleeding time.
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References
- 1 Ruggeri ZM, Zimmerman TS. The complex multimeric composition of factor VUI/von Willebrand Factor. Blood 1981; 57: 1140-1143
- 2 Counts RB, Paskell SL, Elgee SK. Disulfide bonds and quaternary structure of factor VIII/von Willebrand factor. J Clin Invest 1978; 62: 702-709
- 3 Weiss HJ, Turitto VT, Baumgartner HREffect. Shear rate-dependent decrease of adhesion in von Willebrand’s disease and the Bernard-Soulier syndrome. J Lab Clin Med 1978; 92: 750-764
- 4 Baumgartner HR, Tschopp TB, Meyer D. Shear rate dependent inhibition of platelet adhesion and aggregation on collagenous surfaces by antibodies to human factor VUI/von Willebrand factor. Br J Haematol 1980; 44: 127-139
- 6 Ruggeri ZM, Zimmerman TSPlatelets, Weiss HJ, Baumgartner HR. Decreased adhesion of platelets to subendothelium in von Willebrand’s disease. J Lab Clin Med 1974; 83: 296-300
- 7 Turitto VT, Weiss HJ, Zimmerman T, Sussan II. Factor VIH/von Wille- brand factor in subendothelium mediates platelet adhesion. Blood 1985; 65: 823-831
- 8 Ruggeri ZM, De Marco L, Gatti L, Bader R, Montgomery RR. Platelets have more than one binding site for von Willebrand factor. J Clin Invest 1983; 72: 1-12
- 9 Nokes TJ C, Mahmoud NA, Savidge GF, Goodall AH, Meyer D, Edgington TS, Hardisty RM. Von Willebrand factor has more than one binding site for platelets. Thromb Res 1984; 34: 361-366
- 10 Nurden AT, Caen JP. Different glycoprotein abnormalities in thromb- asthenic and Bemard-Soulier platelets. Semin Hematol 1979; 16: 234-250
- 11 Tobelem G, Levy Toledano S, Bredoux R, Michel H, Nurden A, Caen JP, Degos L. New approaches to determination of specific functions of platelet membrane sites. Nature 1976; 263: 427-429
- 12 Fujimura Y, Titani K, Holland LZ, Russel SR, Roberts JR, Elder JH, Ruggeri ZM, Zimmerman TS. A reduced and alkylated 52148 kDa fragment beginning at amino acid residue 449 contains the domain interacting with platelet glycoprotein lb. J Biol Chern 1986; 261: 381-385
- 13 Titani K, Kumar S, Takio K, Ericsson LH, Wade RD, Ashida K, Walsh KA, Chopek MW, Sadler JE, Fujikawa K. Amino acid sequence of human von Willebrand factor. Biochemistry 1986; 25: 3171-3184
- 14 Mohri H, Fujimura Y, Shima M, Yoshioka A, Houghten RA, Ruggeri ZM, Zimmerman TS. Structure of the von Willebrand factor domain interacting with glycoprotein lb. J Biol Chem 1988; 263: 17901-17904
- 15 Coller BS, Peerschke El, Scudder LE, Sullivan CA. Studies with murine monoclonal antibody that abolishes ristocetin-induced binding of von Willebrand factor to platelets. Additional evidence in support of GPIb as a platelet receptor for von Willebrand factor Blood 1983; 61: 99-110
- 16 Howard MA, Perkin J, Salem HH, Firkin BG. The agglutination of human platelets by botrocetin: evidence that botrocetin and ristocetin act at different sites on the factor VIII molecule and platelet membrane. Br J Haematol 1984; 57: 25-35
- 17 Fujimoto T, Hawiger J. Adenosine diphosphate induces binding of von Willebrand factor to human platelets. Nature 1982; 297: 154-156
- 18 Fujimoto T, Ohara S, Hawiger J. Thrombin-induced exposure and prostacyclin inhibition of the receptor for factor VIH/von Willebrand factor on human platelets. J Clin Invest 1982; 69: 1212-1222
- 19 Ruggeri ZM, Bader R, Demarco L. Glanzmann’s thrombasthenia: Deficient binding of von Willebrand factor to thrombin-stimulated platelets. Proc Natl Sci USA 1982; 79: 6038-6041
- 20 Becker HB, Miller JL. Effects of an antiplatelet glycoprotein lb antibody on hemostatic function in the guinea pig. Blood 1989; 74: 690-694
- 21 Read MS, Potter JY, Brinkhous KM. Venom coagglutinin for detection of von Willebrand factor activity in animal plasma. J Lab Clin Med 1983; 104: 74-82
- 22 Sakariassen KS, Nievelstein PF E M, Coller BS, Sixma JJ. The role of platelet membrane glycoproteins lb and IIb-IIIa in platelet adherence to human artery subendothelium. Br J Haematol 1986; 63: 681-691
- 23 Meyer D, Girma JP. Von Willebrand factor: Structure and function. Thromb Haemost 1993; 70: 99-104
- 24 Bemdt MC, Ward CM, Booth WJ, Castaldi PA, Mazurov AV, Andrews RK. Identification of aspartic acid 514 through glutamic acid 542 as a glycoprotein Ib-IX complex receptor recognition of von Willebrand factor by ristocetin and botrocetin. Biochemistry 1992; 31: 11144-11151
- 25 Kupinski JM, Miller J. Synthesis by guinea pig megakaryocytes of platelet glycoprotein receptors for fibrinogen and von Willebrand factor. Thromb Res 1986; 43: 345-352
- 26 Gralnick HR, Williams S, McKeown L, Kramer L, Krutzsch H, Gorecki M, Pinet A, Garfinkel LI. A monomeric von Willebrand factor fragment, Leu-504-Lys-728, inhibits von Willebrand factor interaction with glycoprotein Ib-IX. Proc Natl Acad Sci USA 1992; 89: 7880-7884
- 27 Cisse-Thiam M, Perreault P, Chenu J, Girma JP, Meyer D, Drouet LO. Peptide 692-708 derived from the GPIb-binding domain of von Willebrand factor is a potent inhibitor of in vivo thrombosis in the guinea pig. Thromb Haemost 1991; 65: 1180 (abstract)
- 28 Drouet L, Godard B, Gebrane-Younes J, Pignaud G, Chameroy V. In vivo effects of a dye pulsed laser on microcirculation. In Biology and Pathology of Platelet-Vessel Wall Interactions. Jolles G. (ed) New York: Academic Press Inc; 1987. pp 351-373
- 29 Miller JL, Cisse-Thiam M, Drouet LO. Reduction in thrombus formation by PG-1 F(ab')2, an anti-guinea pig platelet glycoprotein lb monoclonal antibody. Arterioscler Thromb 1991; 11: 1231-1236
- 30 Cook NS, Zerwes H-G, Tapparelli C, Powling M, Singh J, Metternich R, Hagenbach A. Platelet aggregation and fibrinogen binding in human, rhesus monkey, guinea pig, hamster, and rat blood: activation by ADP and a thrombin receptor peptide and inhibition by glycoprotein II/IIIa antagonists. Thromb Haemost 1993; 70: 531-539
- 31 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: 1730-1741
- 32 Moake JL, Turner NA, Stathopoulos NA, Nolasko LH, Heliums JD. Involvement of large plasma von Willebrand Factor (vWF) multimers and unusually large vWF forms derived from endothelial cells in shear stress-induced platelet aggregation. J Clin Invest 1986; 78: 1456-1461
- 33 Ikeda Y, Handa M, Kawano K, Kamata T, Murata M, Araki Y, Anbo H, Kawai Y, Watanabe K, Itagaki I, Sakai K, Ruggeri ZM. The role of von Willebrand factor and fibrinogen in platelet aggregation under varying shear stress. J Clin Invest 1991; 87: 1234-1240
- 34 Dardik R, Ruggeri ZM, Savion N, Gitel S, Martinowitz U, Chu V, Varon D. Platelet aggregation on extracellular matrix. Effect of a recombinant GPIb- binding fragment of von Willebrand factor Thromb Haemost 1993; 70: 522-526