Edward P. Kirby

The influence of Haemaccel, fibrinogen and albumin on ristocetin-induced platelet aggregation. Relevance to the quantitative measurement of the ristocetin cofactor.

Abstract Albumin, fibrinogen and Haemaccel inhibit ristocetin-induced platelet aggregation by binding ristocetin. Increasing the ristocetin concentration can overcome this inhibition. Ristocetin precipitates albumin, fibrinogen, and Haemaccel. Precipitation is dependent on both the protein and the ristocetin concentration. At ristocetin concentrations less than about 2.5 mg/ml fibrinogen seems to be the only plasma protein which is precipitated. The optimal ristocetin concentration to be used for assay of the ristocetin cofactor depends on the type and concentration of proteins in the test system. Ristocetin concentration must be high enough to overcome inhibitory binding, but low enough to prevent fibrinogen precipitation. Conditions are outlined for optimizing the assay of the ristocetin cofactor.

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.

The effects of neuraminidase treatment on the biological activities of factor VIII

Abstract Complete removal of sialic acid from bovine Factor VIII by treatment with neuraminidase results in no loss of platelet agglutinating factor activity or procoagulant activity. There is no alteration in the immunological properties of the molecule, as measured by double immunodiffusion or the Laurell assay, although its electrophoretic mobility is slightly decreased. Removal of sialic acid from human Factor VIII, however, results in a 74 percent loss of ristocetin cofactor activity.

Molecular Mechanisms in Blood Coagulation

Publisher Summary This chapter reviews the molecular mechanisms in blood coagulation. In vivo , the blood platelets play a primary role in blood coagulation in several ways. Often the first response to damage to a blood vessel is aggregation of platelets at the site of injury. They tend to form a hemostatic plug to inhibit further bleeding. Also, certain platelet components are released during this response of platelets to vessel injury. Platelets are known to contain factor XIII, and during coagulation platelets also release substances such as serotonin that result in vasoconstriction. Platelets are also important in the retraction of the fibrin clot. Blood coagulation can be divided into two major pathways, referred to as the intrinsic and extrinsic mechanisms. The intrinsic pathway refers to those reactions that lead to fibrin formation and that utilize only factors present in plasma. The extrinsic pathway is composed of plasma factors and components present in tissue extracts. Both of these pathways play an important physiological role in mammalian hemostasis.

Complete cDNA sequence of bovine α1-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.

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.

Interaction of bovine factor IIa 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.

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.

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 prekailikrein 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 sulfateinduced prekailikrein 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.