Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.27 (thermolysin)
 1,894 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have used monoclonal antibodies (M Abs) and proteolytic fragmentation to localize structurally the functional sites of human von Willebrand factor (vWF) responsible for interaction with membrane glycoproteins GPIb, GPIIb/IIIa, and with collagen. SpII (215 kd) and SpIII (320 kd), the S aureus V-8 protease homodimeric fragments representing the carboxy-terminal and amino-terminal segments of the vWF subunit, competitively inhibited the binding of multimeric vWF to thrombin-stimulated or ristocetin-stimulated platelets, respectively. Specific saturable binding of each fragment was observed to stimulate platelets appropriately and was inhibited only by selected M Abs that both bound to the specific fragment and inhibited the corresponding function. M Ab 9, which blocks thrombin-induced binding of vWF to platelets, inhibited binding of SpII to platelets and bound to SpII as well as to a dimeric, 86-kd thermolysin fragment composed of 42-kd and 23-kd subunits, each possessing the epitope. Binding of SpII was also inhibited by a M Ab to GPIIb/IIIa. Thus, it appears that a portion of the carboxy-terminal end of vWF contains the ligand site for the GPIIb/IIIa receptor. In contrast, M Ab H9, which blocks ristocetin-induced binding of vWF to platelets, inhibited binding of SpIII to platelets and bound to SpIII as well as to monomeric 33-kd and 28-kd subtilisin fragments. Binding of SpIII to platelets was also inhibited by a M Ab to GPIb. Thus, it appears that a small segment of the amino-terminal part of vWF contains the ligand for the platelet GPIb receptor. The collagen binding site of vWF was localized with M Ab B203, which inhibits vWF interaction with collagen. This M Ab also bound to SpIII as well as to monomeric 26-kd and 23-kd subtilisin fragments. Thus, the third functional site responsible for collagen binding appears to be localized on the amino-terminal portion of vWF, in a linear sequence different from those responsible for interaction with either of the platelet receptors. These assignments of functional sites should facilitate the localization of structural defects of vWF in the various forms of vWD and support the role of vWF as an adhesive protein with multiple interactive sites.
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PMID:Mapping of distinct von Willebrand factor domains interacting with platelet GPIb and GPIIb/IIIa and with collagen using monoclonal antibodies. 300 90

A large-scale method for the isolation of von Willebrand factor (vWF) from human factor VIII concentrates was developed in order to study the structure of this protein and its platelet binding activity. vWF is composed of a number of glycoprotein subunits that are linked together by disulfide bonds to form a series of multimers. These multimers appear to contain an even number of subunits of 270K. Two minor components of Mr 140K and 120K were also identified, but these chains appear to result from minor proteolysis. The smallest multimer of vWF contained nearly equimolar amounts of the 270K, 140K, and 120K subunits, while the largest multimers contained less than 20% of the two minor components. Amino acid sequence analysis, amino acid composition, and cleavage by cyanogen bromide indicate that the 270K subunits are identical and each is a single polypeptide chain with an amino-terminal sequence of Ser-Leu-Ser-Cys-Arg-Pro-Pro-Met-Val-Lys and a carboxyl-terminal sequence of Glu-Cys-Lys-Cys-Ser-Pro-Arg-Lys-Cys-Ser-Lys. Platelet binding in the presence of ristocetin was 8-fold greater with multimers larger than five (i.e., containing more than 10 subunits of 270K) as compared to multimers less than three (containing less than six subunits of 270K). However, partially reduced vWF (Mr 500K), regardless of whether it was prepared from large or small molecular weight multimers, gave platelet binding similar to that of the smallest multimers. Likewise, partial proteolysis by elastase, thermolysin, trypsin, or chymotrypsin produced small "multimer-like" proteins with platelet binding properties similar to either partially reduced vWF or to the smallest multimers. We conclude that human vWF contains identical 270K subunits assembled into a multivalent structure. Disassembly by either partial reduction or partial proteolysis produces essentially monovalent protein with platelet binding properties similar to that of the smallest multimers. Multivalency is likely the primary factor responsible for the increase in biological activity with multimer size.
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PMID:Human von Willebrand factor: a multivalent protein composed of identical subunits. 301 99

Elevated levels of lipoprotein(a), which consists of apolipoprotein(a) [apo(a)] covalently linked to a low-density lipoprotein-like moiety, is an independent risk factor for the development of atherosclerosis. We show that a recombinant form of apo(a) [r-apo(a)] binds strongly to fibronectin and fibrinogen, weakly to laminin, and not at all to von Willebrand factor, vitronectin, or collagen type IV. In contrast to the binding of plasminogen to fibronectin, r-apo(a) binding does not appear to be mediated by lysine-dependent interactions, based on the inability of epsilon-aminocaproic acid concentrations up to 0.2 mol/L to significantly decrease r-apo(a) binding to fibronectin. Plasminogen competed weakly for the binding of r-apo(a) to fibronectin, whereas r-apo(a) completely abolished plasminogen binding. The 29- and 38-kd heparin-binding thermolysin fragments of fibronectin, previously identified as the lipoprotein(a) binding domains, were digested with trypsin, and a peptide that retained the ability to bind r-apo(a) was isolated; the sequence of the peptide (AVTTIPAPTDLK) corresponds to the amino terminus of the 29- and 38-kd domains. A synthetic peptide with this sequence was able to compete effectively with fibronectin for r-apo(a) binding.
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PMID:Binding of recombinant apolipoprotein(a) to extracellular matrix proteins. 794 5