Molecular cloning and functional expression of contortrostatin, a homodimeric disintegrin from southern copperhead snake venom.

Contortrostatin is a unique dimeric disintegrin isolated from southern copperhead snake venom. Through antagonism of integrins alphaIIbbeta3, alpha5beta1, alphavbeta3, and alphavbeta5, contortrostatin inhibits platelet aggregation and disrupts cancer cell adhesion and invasion. We cloned cDNA from a library made from the venom gland cells of Agkistrodon contortrix contortrix using polymerase chain reaction. We found that the contortrostatin gene is part of a precursor composed of proprotein, metalloproteinase, and disintegrin domains. The precursor cDNA is 2027 bp with a 1449-bp open reading frame. The disintegrin domain is 195 bp encoding 65 amino acids. Like other members of the disintegrin family, each subunit of contortrostatin has an RGD site, and the cysteine alignment is conserved. The disintegrin domain of the cDNA has been expressed in a eukaryotic expression system as a homodimeric fusion protein with an immunoglobulin. The recombinant protein is recognized by an antiserum against native contortrostatin in Western blot. Both the native and recombinant proteins bind to integrins alphavbeta3 and alphavbeta5. Like native contortrostatin, the recombinant fusion protein inhibits platelet aggregation, blocks cancer cell adhesion to fibronectin and vitronectin, and prevents invasion of cancer cells through a Matrigel barrier. The success of functional expression not only validates the cDNA cloning of this disintegrin, but also provides adequate material for functional studies of contortrostatin.

Contortrostatin, a homodimeric disintegrin, binds to integrin alphavbeta5.

Contortrostatin is a homodimeric disintegrin from snake venom. We have shown that contortrostatin binds to integrins alphaIIbbeta3, alpha5beta1, and alphavbeta3. We now use several criteria to demonstrate the binding of contortrostatin to alphavbeta5. First, incubation of T24 cells, which express alphavbeta3 and alphavbeta5, with antibody against alphavbeta3 failed to completely inhibit adhesion of cells to vitronectin. However, pretreatment of the cells with contortrostatin or the combination of antibodies against alphavbeta3 and alphavbeta5 completely blocked adhesion to vitronectin. By contrast, either anti-alphavbeta5 alone or contortrostatin blocked adhesion of an alphavbeta3-negative T24 subline. Second, contortrostatin as well as anti-alphavbeta5 inhibits invasion of OVCAR-5, which express only alphavbeta5. Third, contortrostatin binds to purified alphavbeta5 in a saturable manner. Finally, radioligand binding assays yielded a K(d) value of 24 nM for [(125)I]contortrostatin binding to alphavbeta5. This investigation identifies alphavbeta5 as a binding site for contortrostatin. Blockage of alphavbeta5 by contortrostatin inhibits alphavbeta5-mediated adhesion and invasion.

Detection of small quantities of double stranded DNA by enhanced fluorescence of substituted aldehyde modified polydeoxynucleotide-ethidium bromide complexes.

A method for the detection of small amounts of double stranded DNA by a simple incubation procedure involving the reaction of the nucleotide bases in DNA with chloroacetaldehyde is described. Following incubation, the presence of DNA may be visualized by the orange fluorescence emitted when ethidium bromide (EtBr) is added. Alternatively, the aldehyde/DNA mixture may be separated by agarose gel electrophoresis and individual double stranded DNA visualized by exposing the gel to ethidium bromide and observing the orange ultraviolet light induced fluorescence of the aldehyde modified double stranded ethidium bromide complex (Waring, M.J. (1965) J. Mol. Biol. 13, 269-282). At least a 20-fold increase in the detection of double stranded DNA is possible by this procedure. An important aspect of this method is that the visualization of single stranded DNA is not enhanced.

5-day storage of human platelet concentrates in 30 ml of plasma or artificial medium.

Optimal conditions for the storage of platelet concentrates were studied by changing 5 environmental parameters: bag composition (PL146 vs. PL732), volume of plasma (60 vs. 30 ml), anticoagulant (CPDA-1 vs. heparin), nutrient (glucose vs. fructose) and medium (plasma vs. artificial medium). A full bilevel factorial study was conducted to evaluate each variable alone and in combination with the other variables for their effects on platelet aggregation and release in response to single and pairs of stimuli. Serotonin uptake, pCO2, platelet count, lactate, glucose, pO2, pH and white blood cell concentration were also measured after 3 and 5 days of storage. Platelets that were stored in PL146 bags had reduced responses to stimulation by 3 days and markedly impaired responses after 5 days relative to platelets that were stored in PL732 bags. There was a large drop in pH and platelet responsiveness when platelets were stored in a volume of 30 ml in PL146 bags; these were not found when platelets were stored in 30 ml in PL732 bags. Replacing plasma with an artificial medium or adding fructose or heparin and calcium to plasma yielded platelets that were equally functional as routine controls in CPD-A1 plasma. It was concluded that replacement of plasma with 60 ml of artificial medium or a reduction of plasma volume with storage in PL732 bags are two possible mechanisms of obtaining more plasma from blood donations without compromising maximum platelet storage life.

Survival and recovery of human platelets stored for five days in a non-plasma medium.

Human blood platelets were stored for five days as concentrates in 60 mL of: (a) plasma; (b) non-plasma medium with anticoagulant; and (c) non-plasma medium without anticoagulant. All preparations were equally functional when tested for platelet aggregation and release reaction in response to single agonist or synergistic pairs of agonists in vitro. Platelets stored in non-plasma medium with anti-coagulant had lower kallikrein, fibrino(gen)peptide A, lactate, and beta-thromboglobulin than did plasma controls after five days. In vivo recovery and survival of platelets stored in non-plasma medium with anticoagulant were 51.2% +/- 4.3% and 8.7 +/- 0.3 days, respectively, which were not statistically different from plasma controls of 39.2% +/- 4.9% and 7.2 +/- 0.8 days, respectively. It is concluded that platelets can be stored for five days in a non-plasma medium and still have good in vivo recoveries and survivals.

Platelet storage in a plasma-free medium.

Currently, platelet concentrates are stored in 50 to 60 ml of plasma. A major drawback to storage in plasma is the considerable loss of platelet function which occurs during storage. A modified Tyrodes medium has been developed for storage of platelets. A comparison between platelet concentrates stored in this medium and in plasma showed that platelet aggregation and release responses to synergistic pairs of stimuli were equivalent for both types of concentrates on the day of preparation and after 72 hours. Platelet aggregation and release responses to single stimuli, the content of membrane glycoproteins, and the pH declined during storage but were similar for both preparations. The data show that plasma is not required to maintain in vitro platelet function during storage of platelet concentrates, but in vivo functions remain to be determined. The use of an artificial medium has the advantages of decreasing patient exposure to plasma contaminants, generating additional plasma for fractionation, and controlling more exactly the storage environment.