The interaction of bovine factor VIII with human platelets.

Treatment of human platelets with purified bovine Factor VIII caused three types of aggregation: (a) primary agglutination; (b) secondary aggregation involving the platelet release reaction; and (c) super-aggregation, in which the platelets were gathered into only a few large clumps. Removal of calcium ions or treatment with p-hydroxymercuiriphenyl sulfonate blocked the release reaction, but not primary agglutination or super-aggregation. Platelets treated with formalin were not aggregated by ADP, thrombin, or collagen, but were agglutinated by bovine Factor VIII, although they did not show super-aggregation. For malin-treated platelets were agglutinated by phytohemagglutinin P less extensively and less rapidly than by bovine Factor VIII. Treatment of platelets and Factor VIII with neuraminidase released 60 and 53%, respectively, of the sialic acid residues without affecting the agglutination reaction or the procoagulant activity of the Factor VIII. Agglutination was inhibited by high salt concentrations, dextran sulfate, and heparin. During agglutination, both the procoagulant and platelet-agglutinating activities of Factor VIII became bound to the platelet surface.

Mechanism of activation of coagulation factor XI by factor XIIa studied with monoclonal antibodies.

The interaction of Factor XIIa with Factor XI was investigated using two monoclonal antibodies, one (3Cl) directed against the heavy chain of Factor XIa and the other (5F4) against its light chain. 3C1 either as intact IgG or as Fab' fragment, enhanced the rate of Factor XIa generation in the fluid phase but inhibited it in the presence of kaolin and high molecular weight (HMW) kininogen. In contrast, the Fab' fragments of 5F4 inhibited only the fluid phase activation and had no effect on the surface-mediated activation. 3C1 was found to block the binding of Factor XI to HMW kininogen, whereas 5F4 did not. We conclude: a domain on the heavy chain region of Factor XI is essential for binding to HMW kininogen and for optimal surface-mediated activation by Factor XIIa; and binding of 3C1 to Factor XI changes its conformation rendering it a more favorable substrate for Factor XIIa in the fluid phase.

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.

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.

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.

A rapid quantitative staphylococcal co-agglutination assay. Utilization for the assay of bovine factor VIII-related antigen.

A simple and rapid technique to measure bovine factor VIII-related antigen has been developed which utilizes protein A-bearing staphylococci and monospecific rabbit antiserum to bovine factor VIII. Staphylococci coated with a specific antibody agglutinate when they are mixed with the specific antigen. We have used an aggregometer to detect an quantitate the agglutination of the antibody-coated staphylococci. The assay has been optimized with respect to amount of antiserum needed for coating staphylococci, concentration of antibody-coated staphylococci, pH and ionic strength of the assay system, and stirring speed of the aggregometer. The staphylococcal co-agglutination assay as monitored by an aggregometer is at least 10 times more sensitive than the conventional slide agglutination method, and can detect as little as 0.1 microgram/ml of factor VIII antigen. It however, cannot be used to quantitate factor VIII-related antigen in plasma, since plasma contains some components which can non-specifically agglutinate staphylococci.

Characterization of a form of bovine factor VIII which does not aggregate human platelets.

Fractionation of partially purified bovine factor VIII on tricalcium citrate columns produces a material which contains high levels of factor VIII procoagulant activity and factor VIII-related antigen, but does not aggregate human platelets. The procoagulant activity can be blocked by human inhibitors of factor VIII: C and by rabbit antibody to bovine factor VIII. Its activity can be increased by thrombin modification. The apparent molecular weight of this form of factor VIII is significantly lower than that of the forms of factor VIII which contain platelet aggregating activity, and it exhibits a higher mobility on agarose electrophoresis.

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.

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.

Kinetic analysis of bovine factor VIII in the hemophilic dog.

Bovine factor VIII, which did not contain platelet aggregating factor activity, was infused into hemophilic dogs. Factor VIII procoagulant (VIII:C) levels in the dogs increased dramatically, then decreased in a biphasic manner. The half-life of the longest component was 3-7 hrs. The infusions were hemostatically effective and also caused a prolonged shortening of the activated partial thromboplastin time. These studies demonstrate that the platelet aggregating factor activity of bovine factor VIII is not essential for its maintenance in the circulation and that preparations lacking this activity may be clinically useful. When concentrates of partially purified factor VIII: C (essentially free of both platelet aggregating factor and factor VIII-related antigen) were infused, marked increases in VIII: C levels were also observed, but the half-life was significantly shorter (T1/2 of approximately 1 hr.).

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.

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.

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.

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.