Collagen suspension for platelet aggregation test

Collagen-induced pletelet aggregation in Born's method is very important in hemostatic tests, and many reports of this test have been presented. There are only few reports which were described about the concentration of collagen. But very high dose of collagen were used in these reports. The reasons are suspected it is difficult to make small particle of collagen fiber and collagen suspension is not so stable.In this report, the method how to make stable collagen suspension containing small sized collagen fiber is presented, and minor changes in collagen-induced platelet aggregation of hemorrhagic patients are detected by using this collagen suspension.One hundred mg of bovine Achilles tendon collagen (Sigma Chem. Co., St. Louis, Mo. U. S. A.) (lot No., 92C-8060, 53C-8140) in immersed in 50ml of 0.03N. HCl and homogenized with “Virtis Homogenizer 45” (Virtis Co., U. S. A., flask; 16-207, blade; 16-108), at 10, 000rpm, for 40 minutes, in ice bath. The homogenized collagen suspension had milky colour and high viscosity. This suspension was stored in plastic tubes and in refrigerator until use. This suspension was diluted in plastic tube with 0.003N. HCl in normal saline, pH was about 3.3. The concentration of collagen was decided by absorbance at O. D. 280nm in spectrophotometer. Scince the diluted collagen suspension which had same value of 100μg/ml of standard bovine serum albumin solution in Lowry's method, showed 0.1 at O. D. 280mμ, so we called the concentration of collagen was 100ug/ml.The final concentration of 2μg/ml this collagen suspension was able to induced platelet aggregation completly. But the final concentration of 3μg/ml of collagen suspension was seemed to be safety range, and the final concentration of this collagen suspension was enough dose to induce platelet aggregation.Two female hemorrhagic patients, only purpura of legs, had normal values in hemostatic tests except slightly prolonged bleeding time, decreased platelet adhesiveness and slight decrease in ADP-induced platelet aggregation, showed remarkable decrease of collagen-induced platelet aggregation in the final concentration of 3μg/ml. However one of these patients was normal in 10μg/ml collagen-induced platelet aggregation.We think this stable and small sized collagen fiber suspension is useful for routine collagen-induced platelet aggregation test in Born's method, because this collagen suspension is homogenious and has high reproducibility, then low concentration of collagen can detect the minor change of platelet aggregability.