Concentration of platelets and growth factors in canine autologous conditioned plasma.

OBJECTIVES: To report the concentration of blood cells and selected growth factors in canine autologous conditioned plasma (ACP). METHODS: The density of blood cells in whole blood (WB), ACP and standard plasma preparation (SP) of 10 healthy mature dogs was determined. In both ACP and SP, the concentration of insulin-like growth factor-1 (IGF-1), epidermal growth factor, vascular endothelial growth factor, platelet-derived growth factor-AA, platelet-derived growth factor-AB, platelet-derived growth factor-BB, transforming growth factor-ß1 (TGF-ß1), and transforming growth factor-ß2 was measured using the ELISA technique. In another ten dogs, ACP was prepared using an ultra-soft spinning protocol, and again blood cell density was compared to that obtained in WB. RESULTS: The density of platelets in ACP was significantly higher than that in SP (p = 0.0002), but there was not any significant difference between ACP and WB, nor between WB and ACP prepared using softer centrifugations. Interestingly, only for IGF-1, PDGF-BB, and TGF-ß1 could reliable measurements be obtained, showing a significant increase in PDGF-BB and TGF-ß1 concentrations in ACP compared to SP (p = 0.001, p = 0.0028). Regarding IGF-1 content, there was not any significant difference between ACP and SP. CLINICAL SIGNIFICANCE: Canine ACP prepared according to the manufacturer's recommendations, or by using a softer spin does not show the same specifications as human ACP, which shows a doubling in platelet count compared to WB. Even though canine ACP has a similar number of platelets per injected volume and consequently, probably the same amount of injected growth factors than WB, application of canine ACP would not be associated with the proinflammatory potential reported for WB, as it is almost free of erythrocytes and nucleated cells.

Nonradical oxidants of the phagocyte type induce the activation of plasmatic single chain- urokinase.

Single chain- urokinase (scu-PA) is the proenzyme of the plasminogen activator urokinase (tcu-PA). In human blood scu-PA is of great stability. Activated phagocytes generate large amounts of single chain- urokinase and of reactive oxidants (chloramines and HOCl). Since these cells participate in physiologic fibrinolysis, we were interested in the interaction between plasmatic scu-PA and chloramines. The oxidants dose dependently induce the activation of plasmatic scu-PA. Optimal activation of scu-PA occurs at about 3-5 mmol/l of chloramine-T. The findings suggest a control mechanism of scu-PA stability/activity by oxidatively modifiable plasma proteins, such as alpha-2-antiplasmin. The oxidation mechanism seems to be mediated by singlet molecular oxygen, an excited oxygen species. Basing on this scu-PA/oxidant synergism a sensitive and fast functional assay of scu-PA in human plasma is presented. Plasmatic inhibitors normally interfering with functional scu-PA measurements are inactivated by addition of chloramine-T, imitating the physiological oxidants generated by activated phagocytes. The scu-PA concentration in plasma of n = 36 healthy individuals has been determined to be 5.8 +/- 1.6 ng/ml. The lower detection limit of plasma scu-PA by the procedure described is about 1.5 ng/ml of plasma. By means of this technique scu-PA concentration during thrombolytic therapy can be measured within minutes in undiluted (direct) plasma samples, allowing adjustments of the scu-PA dosage. The present study gives further credence for a role of singlet molecular oxygen, possibly a new type of locally acting hormones (autacoid), in the regulation of the fibrinolytic pathway.

Reduced O6-methylguanine repair in fibroblast cultures from patients with lung cancer.

The activity of O6-methylguanine-DNA methyltransferase was determined in fibroblast cultures from 45 patients with lung cancer, 39 patients with cutaneous malignant melanoma, and 29 healthy controls. This enzyme is a critical parameter for the capacity to repair O6-methylguanine (O6-mGua) adducts in DNA, and a decreased activity might therefore be responsible for an enhanced susceptibility to cancer. The assay was performed with 8 x 10(6) fibroblasts which were homogenized and incubated with a known amount of O6-mGua containing DNA. The remaining substrate was determined fluorimetrically after high performance liquid chromatographic separation. O6-mGua repair was significantly reduced in lung cancer patients [6.64 +/- 4.32 (SD) pmol O6-methylguanine repaired/8 x 10(6) cells] as compared to healthy controls [10.35 +/- 5.42, P less than 0.0022] or patients with cutaneous malignant melanoma [10.83 +/- 6.66]. The lowest mean values were detected in a subgroup of 16 lung cancer patients with a tumor manifestation below 46 years of age (5.06 +/- 3.89). Fibroblasts from 4 patients with lung cancer had no detectable repair. We conclude that a reduced capacity to remove O6-mGua adducts may represent a further mechanism of individually enhanced lung cancer risk.