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Proteolytic studies on the structure of bovine von Willebrand factor
1Department of Biochemistry, Temple University Health Science Center, Philadelphia, Pennsylvania 19140.
Abstract:
Bovine von Willebrand factor (vWF) was digested with protease I (P-I), a metalloprotease isolated from rattlesnake venom. Digestion of vWF for 24 h with P-I yielded a terminal digest consisting of an equimolar mixture of two major fragments (apparent Mr 250K and 200K). The 250-kilodalton (kDa) fragment consists of a 125-kDa chain from one subunit and a 45- and 78-kDa polypeptide chain from an adjacent subunit. The 200-kDa fragment consists of a 97-kDa chain from one subunit and a 35- and 61-kDa polypeptide chain from an adjacent subunit. The 200-kDa fragment binds to heparin, and the heparin binding domain is located on the 97-kDa polypeptide chain. This fragment also competes with labeled, native vWF for binding to formalin-fixed human platelets, with an IC50 of 12.5 micrograms/mL (65 nM). However, native vWF has an IC50 of 2.5 micrograms/mL, indicating that the affinity of the 200-kDa fragment for platelets is approximately one-fifth that of vWF. The 200-kDa fragment agglutinates platelets, but its agglutinating ability is only 5% that of the native molecule. Only the 200-kDa fragment is recognized by monoclonal antibodies 2 and H-9, which are directed against vWF and inhibit vWF binding to platelet glycoprotein Ib (GPIb). Immunological studies, using nine monoclonal antibodies directed against vWF, and the demonstration that the heparin and GPIb binding domains are located on only one fragment suggest that the two fragments are composed of different regions of the vWF subunit. Analysis of the P-I cleavage pattern suggests that all vWF subunits are not cleaved in the same fashion. The first cleavage on half of the subunits generates the 45-kDa terminal and 175-kDa intermediate digest products. The 175-kDa chain is again cleaved, producing the 97- and 78-kDa terminal polypeptide chains. However, the first cleavage of the other subunits generates the 35-kDa terminal and the 186-kDa intermediate digest product, which upon cleavage produces the 125- and 61-kDa terminal polypeptide chains. Immunological data support the asymmetric cleavage pattern. An epitope for a monoclonal antibody is present on both the 186- and 175-kDa intermediate digest products but is only found on one terminal digest fragment, the 78-kDa polypeptide chain, suggesting that the 186- and 175-kDa polypeptides are cleaved at different sites.(ABSTRACT TRUNCATED AT 400 WORDS)
Insights
Rattlesnake venom protease I (P-I) digests bovine von Willebrand factor (vWF) into two fragments. The 200-kDa fragment retains heparin and platelet binding capabilities, though with reduced affinity compared to native vWF.
Area of Science:
- Biochemistry
- Proteomics
- Venom research
Background:
- Von Willebrand factor (vWF) is a large multimeric glycoprotein crucial for primary hemostasis.
- Understanding vWF structure and function is vital for diagnosing and treating bleeding disorders.
Purpose of the Study:
- To investigate the proteolytic cleavage of bovine vWF by protease I (P-I) from rattlesnake venom.
- To characterize the functional properties of the resulting vWF fragments.
Main Methods:
- Digestion of bovine vWF with purified protease I (P-I).
- Analysis of digestion products using SDS-PAGE to determine fragment sizes.
- Heparin binding assays.
- Platelet aggregation assays.
- Competition assays using radiolabeled vWF and monoclonal antibodies.
Main Results:
- Protease I generated an equimolar mixture of 250-kDa and 200-kDa vWF fragments.
- The 200-kDa fragment exhibited heparin-binding activity, localized to a 97-kDa polypeptide.
- This fragment also bound to human platelets via glycoprotein Ib (GPIb), with approximately one-fifth the affinity of native vWF.
- The 200-kDa fragment induced platelet agglutination, albeit at 5% the efficacy of native vWF.
- Monoclonal antibodies recognizing vWF binding domains only recognized the 200-kDa fragment.
Conclusions:
- Protease I cleaves vWF into distinct functional fragments.
- The 200-kDa fragment contains both heparin and GPIb binding domains, suggesting it represents specific regions of the vWF subunit.
- vWF subunits undergo asymmetric cleavage by P-I, leading to heterogeneous fragment generation.