Immunologic studies of native and modified human factor VIII/von Willebrand factor

Area of Science:

• Hematology
• Immunology
• Biochemistry

Background:

• Factor VIII/von Willebrand factor (FVIII/vWF) is a large glycoprotein complex.
• It possesses two distinct activities: FVIII procoagulant activity and vWF activity.
• Proteolytic enzymes rapidly inactivate FVIII procoagulant activity while minimally affecting vWF activity.

Purpose of the Study:

• To investigate the structural features of FVIII/vWF responsible for its distinct activities.
• To understand the immunologic properties of FVIII/vWF after proteolytic modification.

Main Methods:

• Generation of rabbit antisera against native, thrombin-inactivated, and plasmin-inactivated FVIII/vWF.
• Assays for FVIII procoagulant activity and vWF activity inhibition.
• Polyacrylamide gel electrophoresis in SDS-urea to analyze protein structure.

Main Results:

• All antisera cross-reacted with modified FVIII/vWF and inhibited a human inhibitor of native FVIII/vWF.
• Antisera potently inhibited vWF activity, but were less potent inhibitors of FVIII activity.
• Antibodies to thrombin-inactivated FVIII/vWF showed similar FVIII inhibitory capacity as antibodies to native FVIII/vWF.
• Antibodies to plasmin-inactivated FVIII/vWF retained significant FVIII inhibitory capacity despite extensive degradation.

Conclusions:

• FVIII/vWF retains immunologic properties after significant proteolytic degradation.
• The conformation of native and proteolytically degraded FVIII/vWF appears similar under nondenaturing conditions.
• Variations in antibody inhibition may stem from antigen degradation during preparation.