Primary binding domain of bovine von Willebrand factor fragment expressed in E. coli.

Bovine vWF cDNA has been cloned from a bovine endothelial cell library. A fragment of this cDNA, corresponding to amino acid sequence Leu 469-Ser 723, called primary adhesion domain (PAD-1), and containing the binding sites for platelet glycoprotein Ib (GPIb), heparin and collagen, has been expressed in E. coli. The reduced and alkylated form of fragment PAD-1 inhibited native vWF binding to GPIb. Fragment PAD-1 bound to heparin and botrocetin in a specific and dose dependent manner as did the native vWF. In a solid-phase assay, fragment PAD-1 bound to calf skin collagen in contrast to a human vWF recombinant fragment (Ser 445-Val 733) which was inactive in the same assay. The studies presented in this paper demonstrated that the A1 domain of bovine vWF contained the GPIb, heparin, botrocetin as well as collagen binding sites and that integrity of the disulfide bond (Cys 509-Cys 695), did not seem to be essential for binding of bovine vWF fragment to GPIb.

Interaction of echicetin with a high affinity thrombin binding site on platelet glycoprotein GPIb.

Echicetin, a protein isolated from Echis carinatus snake venom, inhibited platelet aggregation and secretion induced by low concentrations of thrombin ( < 0.2 U/ml), by binding to platelet glycoprotein Ib (GPIb). The inhibition was not observed when the platelets were stimulated with higher concentrations of thrombin ( > 0.2 U/ml). Echicetin competed with thrombin for binding to the high affinity site on GPIb. Thrombin also inhibited 50% of the binding of 125I-echicetin to the platelets.

ASP514 within the A1 domain of bovine von Willebrand factor is required for interaction with platelet glycoprotein Ib.

A mutant PAD-1 (D514-->Q) of the recombinant fragment PAD-1 comprising Leu469-Ser723 of the A1 domain of bovine von Willebrand factor (vWF) neither inhibited the binding of [125I]vWF to platelets nor the agglutination of human platelets induced by bovine vWF. PAD-1, on the other hand, inhibited human platelet agglutination induced by bovine vWF and [125I]vWF binding to human platelets. Collagen binding properties of the mutant, however, were indistinguishable from those of PAD-1. These results suggested that Asp514 within the A1 domain of vWF is required for interaction of bovine vWF with GPIb receptor on human platelets.

Isolation and characterization of two protease inhibitors from bovine plasma.

Two protease inhibitors (Inh2 and Inh3) from bovine plasma have been isolated and characterized. The apparent molecular weights of the two proteins are 56 and 58 kDa, respectively. Although Inh2 and Inh3 both inhibit trypsin and human neutrophil elastase, only Inh3 is a good inhibitor of chymotrypsin and cathepsin G. Inh3 is much more sensitive to oxidation than Inh2. One murine monoclonal antibody recognizes Inh3 but not Inh2. Inh3 resembles human alpha 1-antitrypsin both structurally and functionally. Inh2, on the other hand, has some structural homology to human alpha 1-antichymotrypsin, but its specificity does not correspond to that of either human alpha 1-antitrypsin or human alpha 1-antichymotrypsin.

PCR to identify specific clones of interest for DNA sequencing.

We describe a rapid method for identifying specific clones of interest for the purpose of sequencing. The method essentially is polymerase chain reaction using one internal primer and one vector specific primer. The procedure is particularly useful when relatively large numbers of clones are to be examined either to establish the nucleotide sequence of a full-length cDNA or to find a specific section of a large DNA. The relative orientations of inserts in different clones can also be determined using the same procedure.

Echicetin: a snake venom protein that inhibits binding of von Willebrand factor and alboaggregins to platelet glycoprotein Ib.

Echicetin, a new protein isolated from Echis carinatus venom by reverse phase and ion exchange chromatography specifically inhibited agglutination of fixed platelets induced by several platelet glycoprotein Ib (GPIb) agonists, such as bovine von Willebrand factor (vWF), alboaggregins, and human vWF in the presence of botrocetin. Unlike alboaggregins, echicetin bound to GPIb but did not induce agglutination of washed or fixed platelets. In contrast to disintegrins, it did not block adenosine 5'-diphosphate (ADP)-induced platelet aggregation in the presence of fibrinogen. The apparent molecular weight of echicetin measured on sodium dodecyl sulfate (SDS) gel electrophoresis was 26 Kd under nonreducing conditions. On reduction, echicetin showed 16 and 14-Kd subunits suggesting that the molecule is a dimer. Reduced echicetin retained its binding activity and its inhibitory effect on the agglutination of fixed platelets induced by bovine vWF. 125I-echicetin bound to fixed platelets with high affinity (kd = 30 +/- 1.8 nmol/L) at 45,000 +/- 2,400 binding sites per platelet. The binding was selectively inhibited by a monoclonal antibody to the 45-Kd N-terminal domain of platelet GPIb, but not by monoclonal antibodies to other regions on GPIb. Binding of 125I-bovine vWF to fixed platelets was strongly inhibited by echicetin. In contrast, bovine vWF showed a much weaker inhibitory activity on binding of 125I-echicetin to platelets. The half life of echicetin in blood was approximately 170 minutes with no detectable degradation. Echicetin significantly prolonged the bleeding time of mice, suggesting that it may inhibit vWF binding to GPIb in vivo as well as in vitro.

Sequencing of the primary adhesion domain of bovine von Willebrand factor.

A cDNA library, constructed from bovine heart endothelial cell poly(A)+ RNA, was screened using a BstXI fragment of human von Willebrand and factor (vWF) cDNA as a probe. This probe codes for the major adhesion domain of vWF that includes the GPIb, collagen and heparin binding domains. Of the ten positive clones obtained, a clone that spanned the region of interest was sequenced by the dideoxynucleotide method yielding a sequence of 1550 bp. This region of the bovine cDNA codes for amino acids corresponding to #262 to #777 in human vWF and encompasses the entire pro adhesion domain. Both the nucleotide sequence and the deduced amino acid sequence are 82% homologous to those of human vWF. Cysteine residues #471, 474, 509 and 695, which form intrachain bonds in human vWF, are also present in the bovine vWF sequence.

Inhibition of prekallikrein activation in human plasma by components of bovine plasma.

Contact of plasma with a negatively charged surface activates prekallikrein and factor XII reciprocally. Activation of prekallikrein by several activators was impaired in bovine plasma when compared to that in human plasma. The activated partial thromboplastin time of bovine plasma, induced by several activators, was significantly longer than that of human plasma. Cleavage of [125I]factor XII was optimum at 10 min in human plasma but took up to 60 min in bovine plasma. Addition of bovine plasma to human plasma caused significant inhibition of dextran sulfate-induced prekallikrein activation, indicating that the impaired rate of contact activation in bovine plasma is due to the presence of inhibitors. The inhibitory effect was greater at lower concentrations of dextran sulfate but could not be abolished by increasing the concentration. The inhibitory activity eluted in two peaks at low and medium salt concentrations on carboxymethyl ion-exchange chromatography of bovine plasma.

Characterization of three alboaggregins purified from Trimeresurus albolabris venom.

Alboaggregins (AL-A, AL-B, AL-C) isolated from Trimeresurus albolabris snake venom represent a new family of proteins which bind to platelet glycoprotein Ib (GPIb). These alboaggregins were purified to homogeneity with ion exchange HPLC (Mono-Q column) and hydrophobic HPLC (TSK Phenyl-5PW column). On SDS-polyacrylamide gel electrophoresis, apparent molecular weights of AL-A, AL-B and AL-C were 52 kDa, 26 kDa, and 121 kDa respectively, under nonreducing conditions. Upon reduction, each alboaggregin showed two types of chains with apparent molecular weights in the range of 15-20 kDa. All three alboaggregins agglutinated formalin-fixed platelets. Agglutination activities and binding of labeled alboaggregins to GPIb were specifically inhibited by the monoclonal antibody AK2 which is directed against the 45 kDa N-terminal region on GPIb, but not by monoclonal antibodies against other epitopes on GPIb. 125I-alboaggregin binding to platelets was not altered by the presence of thrombin. Alboaggregins did not bind to GPIIb/IIIa. Alboaggregins were competitive inhibitors for 125I-bovine vWF binding to platelets. Mutual competition studies between AL-A, AL-B and AL-C for the binding of labeled bovine vWF and AL-B to platelets demonstrated that AL-B and AL-C had a significantly higher affinity than AL-A.

Complete cDNA sequence of bovine alpha 1-antitrypsin.

A cDNA clone coding for the entire bovine alpha 1-antitrypsin molecule has been isolated from a lambda gt11 bovine liver cDNA library using a human alpha 1-antitrypsin cDNA as a probe. The bovine cDNA was sequenced by the dideoxynucleotide chain termination method. Comparison of the translated amino acid sequence of the bovine alpha 1-antitrypsin with those of the human, baboon, sheep, rat and mouse demonstrates the preservation of most of the critical structural determinants. The bovine and the sheep molecules have a sequence homology of 94% and both the molecules contain four cysteine residues; there is only one cysteine in the others.

Alboaggregin-B: a new platelet agonist that binds to platelet membrane glycoprotein Ib.

A new protein, called alboaggregin-B (AL-B), has been isolated from Trimeresurus albolabris venom by ion-exchange chromatography. It agglutinated platelets without the need for Ca2+ or any other cofactor. The purified protein showed an apparent molecular mass on SDS-PAGE and gel filtration of about 23 kDa under nonreducing conditions. Ristocetin did not alter the binding of AL-B to platelets or affect AL-B-induced platelet agglutination. Agglutinating activity was not dependent on either proteolytic or lectin-like activity in AL-B. Binding analysis showed that AL-B bound to platelets with high affinity (Kd = 13.6 +/- 9.3 nM) at approximately 30,800 +/- 14,300 binding sites per platelet. AL-B inhibited the binding of labeled bovine von Willebrand factor (vWF) to platelets. Monoclonal antibodies against the 45-kDa N-terminal domain of platelet glycoprotein Ib inhibited the binding both of AL-B and of bovine vWF to platelets, and also inhibited platelet agglutination induced by AL-B and bovine vWF. Specific removal of the N-terminal domain of GPIb by treatment of the platelets with elastase or Serratia marcescens protease reduced the binding of labeled AL-B and bovine vWF to platelets and blocked platelet agglutination caused by both agonists. Monoclonal antibodies to glycoprotein IIb/IIIa, to bovine vWF, and to bovine serum albumin did not show any effect on the binding of AL-B to platelets. Our results indicate that the binding domain for AL-B on platelet GPIb is close to or identical with the one for vWF. This new protein may be a very useful tool for studying the interaction between platelets and vWF.

Proteolytic and immunologic comparison of human and bovine von Willebrand factor.

The structures of bovine and human vWF were compared by proteolysis with Staphylococcus aureus V8 protease and rattlesnake venom Protease I. Fragments were analyzed for chain composition, heparin binding, collagen binding, platelet agglutinating activity and recognition by a panel of monoclonal antibodies which reacted with both bovine and human vWF. Similar large fragments from the C-terminal domain of vWF were seen in each case. The N-terminal domain resulting from cleavage of bovine vWF was much smaller than that seen upon digestion of human vWF with V8 protease. Protease I destroyed the heparin binding domain in human vWF. Bovine vWF was much less sensitive to proteolysis than was human vWF.

Effect of platelet activation on the agglutination of platelets by von Willebrand factor.

Agglutination of human platelets by bovine von Willebrand factor (vWF) or by human vWF in the presence of ristocetin is inhibited by ADP and by several other platelet agonists but not by epinephrine. Vincristine, which causes a shape change by disrupting microtubules, neither inhibited agglutination nor blocked the effect of ADP. The action of ADP was blocked by ATP, by p-fluorosulfonylbenzoyladenosine, and by the thiol-reactive regents cytochalasin A and p-chloromercuribenzenesulfonate. In contrast to its effects on vWF, ADP enhanced agglutination induced by wheat germ lectin. ADP caused a small decrease in the number and affinity of binding sites for vWF on platelets, too small to explain the inhibition of agglutination. The ability of ADP and other agonists to inhibit agglutination appears to be related neither to inhibition of adenylate cyclase nor to the loss of their discoid shape but rather to the membrane changes that accompany the shape change.

Separation of different receptor-mediated effects of a prostaglandin H2 analogue (U46619) on human platelets by means of human granulocytic elastase and chymotrypsin.

Previous investigations indicated two classes of thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors on human platelets and suggested that shape change and myosin light chain phosphorylation correlated with the occupancy of high affinity receptors while serotonin release was related to a putative low affinity binding component (Morinelli TA et al., Am J Physiol 253: H1035-H1043, 1987). The current study shows that chymotrypsin destroyed three receptor-mediated responses of platelets to U46619 (a TXA2/PGH2 agonist), i.e. shape change, myosin light chain phosphorylation and serotonin release. Human granulocyte elastase selectively inactivated platelet ability to release serotonin following stimulation with U46619, but it did not affect significantly shape change and myosin light chain phosphorylation. In conclusion, it is possible to separate different receptor-mediated effects of U46619 on human platelets by means of human granulocytic elastase and chymotrypsin.

Trigramin: primary structure and its inhibition of von Willebrand factor binding to glycoprotein IIb/IIIa complex on human platelets.

Trigramin, a naturally occurring peptide purified from Trimeresurus gramineus (T. stejnegeri formosensis) snake venom, inhibits platelet aggregation and the binding of 125I-fibrinogen to ADP-stimulated platelets (Ki = 2 X 10(-8) M) without affecting the platelet-release reaction. 125I-trigramin binds to ADP-stimulated and to chymotrypsin-treated normal platelets but not to thrombasthenic platelets. 125I-trigramin binding to platelets is blocked by monoclonal antibodies directed against the glycoprotein IIb/IIIa complex and by Arg-Gly-Asp-Ser (RGDS) [Huang et al. (1987) J. Biol. Chem. 262, 161]. We determined the primary structure of trigramin, which is composed of a single polypeptide chain of 72 amino acid residues and six disulfide bridges. The molecular weight of trigramin calculated on the basis of amino acid sequence was 7500, and the average pI was 5.61. An RGD sequence appeared in the carboxy-terminal domain of trigramin. An amino-terminal fragment (7-33) of trigramin showed 39% homology with a region (1555-1581) of von Willebrand factor (vWF). Trigramin also showed 36% identity in a 42 amino acid overlap and 53% identity in a 15 amino acid overlap when compared with two adhesive proteins, collagen alpha 1 (I) and laminin B1, respectively. Trigramin blocked binding of human vWF to the glycoprotein IIb/IIIa complex in thrombin-activated platelets in a dose-dependent manner. Reduction of trigramin resulted in a marked decrease in its ability to block vWF binding to human platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

Proteolytic studies on the structure of bovine von Willebrand factor.

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)

Interaction of bovine factor XIIa with an inhibitor from bovine plasma.

An inhibitor of factor XIIa has been purified from bovine plasma and characterized (Thornton, R.D. and Kirby, E.P. (1987) J. Biol. Chem. 262, 12714-12721). This inhibitor interacts with XIIa to form a very stable complex with a 1:1 stoichiometry. The active site of XIIa, located on the light chain, is directly involved in the interaction, and complex formation between factor XIIa inhibitor and XIIa can be blocked by diisopropyl fluorophosphate, corn trypsin inhibitor, or the chromogenic substrate S2302. Incubation of the complex with excess XIIa does not result in cleavage of the complex. The complex does not spontaneously dissociate and is stable to boiling, SDS, thiocyanate, acid, and hydroxylamine or Tris at pH 7-10. In addition to complex formation, a cleaved form of factor XIIa inhibitor can be observed. We suggest that the inhibitor is acting as a mechanism-based inactivator, using the criteria of time-dependent inactivation under pseudo-first-order conditions, 1:1 stoichiometry, active site involvement, kinetic protection by substrate or by an active site inhibitor, and partitioning between cleavage of factor XIIa inhibitor and inactivation by complex formation.

Isolation and characterization of an inhibitor of factor XIIa from bovine plasma.

An inhibitor of factor XIIa has been purified to homogeneity from bovine plasma. The purification steps included precipitation of contaminating proteins with polyethylene glycol and chromatography on DEAE-cellulose, Affi-Gel blue, and immobilized wheat germ lectin. The apparent molecular weight of the XIIa inhibitor (called INH1) was 85,000, reduced, and 92,000, nonreduced, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The extinction coefficient (E0.1%(280)) of INH1 is 1.3, and the protein contains 17.7% carbohydrate. Purified antibody to INH1 raised in either rabbits or chickens formed a precipitin line of identity with purified INH1 and a component of bovine plasma, but there was no reaction with purified human inhibitors or with any component of human plasma. INH1 inhibits bovine and human XIIa, bovine and human C1-esterase, and human kallikrein, but does not inhibit bovine kallikrein, bovine trypsin, human plasmin, or human thrombin. This activity is similar to that of C1-inhibitor but different from antithrombin III, alpha 2-antiplasmin, or alpha 1-protease inhibitor. INH1 at a physiological concentration (0.47 microM) causes rapid inactivation of XIIa. The two molecules react in a 1:1 stoichiometry with a second-order rate constant of 1.23 X 10(6) M-1 min-1.

Modification of the platelet-binding domain of von Willebrand factor.

Radioiodinated Bolton-Hunter reagent was used at low specific activity to probe for the function and reactivity of amino groups on von Willebrand factor (vWF), a plasma protein involved in platelet responses to damaged endothelial surfaces. The platelet receptor for vWF contains a membrane protein termed glycoprotein Ib. Modification of only one or two amino groups per vWF subunit caused a 50% reduction in the platelet-agglutinating activity of vWF, and a decrease in its ability to bind to platelets. All multimeric forms of vWF are modified. Loss of platelet-agglutinating activity on modification of less than 2% of the amino groups on each vWF subunit suggests that the amino groups in the glycoprotein Ib-binding domain of vWF are both particularly reactive and essential for its function.