Platelet glycoprotein Ib alpha fusion polypeptides and methods of use thereof
a technology of platelet glycoprotein and fusion polypeptide, which is applied in the direction of peptides, drug compositions, cardiovascular disorders, etc., can solve the problems of further undesirable progression of vascular diseases, and achieve the effect of inhibiting the adhesion of platelets
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example 1
Production and Purification of Recombinant GP1B-IG Gusion Proteins
[0091] Three GP1b-Ig fusion proteins, GP1b302-Ig (SEQ ID NO: 1), GP1b290 Ig (SEQ ID NO:4), and GP1b290 / 2V-Ig (SEQ ID NO:5), were produced by recombinant methods and purified. Chinese hamster ovary (CHO) cells lacking dihydrofolate reductase (DHFR) activity were stability transfected with linearized plasmid DNA consisting of a mammalian expression vector directing the transcription of a GP1b-Ig coding regions in polycistronic fashion with a DHFR selectable maker gene. Candidate expressing cells were selected in medium containing increasing concentrations of methotrexate (MTX) essentially as described in Kaufman et al. Nucleic Acids Res. (1991 )19:4485-90. For collection of GP1b-Ig conditioned medium, CHO cells were grown to near confluent levels on 5-20 culture dishes (150 mm diameter), the cell monolayer was washed twice with PBS and cells were cultured for approximately 24 hrs in medium lacking fetal bovine serum. T...
example 2
In Vitro Inhibition of Platelet Aggregation
[0094] The ability of the glycoprotein Ibα polypeptide-immunoglobulin fusion polypeptide to inhibit platelet aggregation in vitro, was determined. Platelet rich plasma (PRP) from freshly drawn, citrate blood was prepared by differential centrifugation for 10 minutes at 900 rpm. 0.4 mls of PRP (3×108 / ml) was preincubated for 5 minutes at 37° C. with various concentrations of GP1b290 / 2v-Ig. Ristocetin was added to 1.5 mg / ml to induce platelet aggregation. Aggregation was measured using a Sienco DP247E aggregometer. Aggregation was quantified and recorded on a chart recorder by monitoring the increase in light transmittance with stirring at 1000 rpm. As illustrated in FIG. 4, GP1b290 / 2v-Ig inhibited ristocetin induced platelet aggregation.
example 3
In Vivo Inhibition of Repetitive Coronary Artery Thrombosis
[0095] The ability of a glycoprotein Ibα GPIb290 / 2V-Ig polypeptide-immunoglobulin fusion polypeptide to inhibit coronary artery thrombosis in vivo was determined using the procedure described by Folts at al., Circulation 54:365-70, 1976.
[0096] Mongrel dogs, weighing 20-25 kg, were anesthetized with sodium pentobarbital (30 mg / kg i.v.), then intubated and ventilated with room air using a respirator. Venous and arterial catheters were placed. The heart was approached by left thoracotomy through the fifth intercostal space. The pericardium was opened and sutured to the wound edges to provide a cradle without displacing the heart. About 2cm of the left circumflex coronary artery (LCX) was isolated. Mean and dynamic LCX flow was continuously monitored using a perivascular ultrasonic flow probe placed proximally on the artery. After a stabilization period, the endothelium of the LCX was injured by squeezing with a hemostat. A pl...
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