Diagnostic ultrasound activates pure prekallikrein.

Diagnostic ultrasound activates the contact phase of human coagulation. This has been seen in human blood or plasma or with purified factor 12. The present work aimed to quantify a possibly triggering action of ultrasound on purified prekallikrein, the second of the two main triggers of the intrinsic hemostasis cascade. Either 2.7 µg/ml human prekallikrein or for control 1 µg/ml kallikrein in 26% glycerol - 0.54% NaCl-10.6 mmol/l Na3 citrate pH 7.4, in emptied polypropylene coagulation monovettes (Sarstedt) were exposed to diagnostic ultrasound (Siemens Acouson Antares, 5 MHz, 0.6 TIB, 0.6 TIS) for 0-5 min at room temperature (RT). Fifty microliter samples were withdrawn in duplicate and placed into an U-wells high quality microtiter plate (Brand 781600). Then 10 µl 2 mmol/l chromogenic substrate HD-CHG-Ala-Arg-pNA in 0.45% NaCl were added, and the increase in absorbance with time (ΔA405 nm /t at 37°C) was determined by a microtiterplate photometer with a 1 mA resolution (PHOmo; anthos). Exposure to diagnostic ultrasound biphasically increased the chromogenic activity of a prekallikrein solution in 26% glycerol. About 3-4 min ultrasound at 23 °C generated about 0.02 µg/ml kallikrein, that means that about 1% of pure prekallikrein in glycerol was converted into kallikrein. Thus, diagnostic ultrasound activates purified human prekallikrein to kallikrein. The ultrasound energy seems to fold the latent proenzyme prekallikrein into the active enzyme kallikrein. This contributes to explain the triggering action of ultrasound on the contact system of plasmatic human coagulation. Conversion of only 1% of prekallikrein into kallikrein is absolutely sufficient to start the intrinsic coagulation cascade. The clinical consequence of this action of ultrasound on intrinsic coagulation is that patients at risk for thrombosis, for example, patients with insufficiencies of hepatocytes, AT-3, C1-ina, or fibrinolysis should be protected by low-molecular-weight-heparin prior to the exposure of ultrasound, especially upon its prolonged exposure.

The ultrasound frequency determines the degree of intrinsic coagulation activation.

Diagnostic ultrasound activates intrinsic coagulation. The aim of the present work was to quantify the action of different ultrasound frequencies on the contact phase of human blood coagulation. Pooled normal citrated platelet-poor plasma in 2 ml aliquots in polypropylene monovettes was exposed to diagnostic ultrasound, changing the ultrasound frequency from 17 to 15 to 12 to 8 to 7 MHz (at an intensity of 1.1 MI). After 0-2 min (23°C), 400 µl samples were withdrawn and placed into polypropylene Eppendorf cups. Forty microliters of plasma sample was pipetted into U-wells polystyrene microtiter plates of high purity (Brand781600). Immediately thereafter, the recalcified coagulation activity assay (RECA) was performed. Seventeen megahertz ultrasound exposure was the weakest activator of intrinsic coagulation of all frequencies tested: even 2 min of exposure at 23°C enhanced F2a generation by only about three-fold. The shorter the ultrasound exposure, the better the action against intrinsic hemostasis: 0.5 min of ultrasound exposure at 23°C induced less than two-fold thrombin generation in all frequencies tested. One minute ultrasound exposure (23°C) triggered intrinsic coagulation strongest at 8 MHz, showing an approximately four-fold increase in F2a generation. A 1.5 min of ultrasound exposure (23°C) triggered coagulation strongest at 7 MHz, showing an approximately 14-fold increase in F2a generation. Two minute of ultrasound exposure (23°C) triggered coagulation strongest at 15 MHz, showing an approximately 18-fold increase in F2a generation. Ultrasound has to be considered as a potential inducer of pathologic systemic coagulation. Patients at risk for increased coagulation activation and/or liver insufficiency should be protected with low molecular weight heparin, if a prolonged ultrasound diagnostic is planned. Ultrasound frequencies of about 17 MHz are the weakest activators of intrinsic coagulation. Ultrasound frequencies of about 7 MHz might be used for therapeutic induction of coagulation activation, such as in patients with severe cerebral or hepatic hemorrhages.

Regulation of hemostasis by singlet-oxygen (1DeltaO2*).

Hemostasis is the system of generation and destruction of thrombi. It consists of coagulation and thrombolysis and has a plasmatic part and a cellular one, the latter being the thrombocytes and endothelial cells for coagulation and the polymorphonuclear granulocytes (PMN) for thrombolysis. Main products of PMN are oxidants of the hypochlorite/chloramine-type that can generate the nonradical excited oxidant singlet molecular oxygen ((1)DeltaO(2)(*)). Physiologically, (1)DeltaO(2)(*) reacts with methionine and cysteine residues and with carbenic structures in lipids, generating dioxetanes, which upon disruption emit photons in the blue spectrum of light (380-450 nm). It modifies some important hemostasis components in blood: (1)DeltaO(2)(*) inactivates the factors I (fibrinogen), V, VIII, vWF, X, plasminogen activator inhibitor-1 (PAI-1), and alpha2-antiplasmin. (1)DeltaO(2)(*) oxidation of plasminogen and fibrin facilitates their specific cleavage by plasminogen activators and plasmin. Furthermore,(1)DeltaO(2)(*)downregulates thrombocyte-function and upregulates PMN-function. Chloramines seem to be the main physiologic generators of (1)DeltaO(2)(*): in concentrations of 0.1-2 mM in blood they strongly inhibit coagulation and enhance thrombolysis. The biogenesis and reaction pattern of (1)DeltaO(2)(*) is of importance to understand the PMN-physiology in hemostasis, giving rise to new therapy forms of thromboatherothrombosis in man.

Point of care: diagnostics in hemostasis--the wrong direction?

Point-of-care-testing (POCT) is performance of a laboratory assay outside the laboratory by nontrained personnel. The advantages of POCT are: more rapid medical decisions, avoidance of long sample transports, and small samples. The disadvantages of POCT are: no laboratory personnel, insufficient calibration, quality control and maintenance, poor documentation, high costs, difficult comparability POCT/central laboratory. Therefore, disposing of a 24-hour central laboratory, the POCT spectrum should be limited to the vital parameters: K+, Ca++, Na+, glucose, creatinine, blood gases, hemoglobin or hematocrit, NH3, lactate. POCT offers no advantages, if the hospital has a rapid transport system such as a pneumatic delivery to the central laboratory. The rapid diagnosis of the acute hemostasis state of a patient should be performed in the 24-hour central laboratory that is connected to all hospital wards via a good pneumatic delivery.

Singlet oxygen (1O2)-oxidazable lipids in the HIV membrane, new targets for AIDS therapy?

Human immunodeficiency virus (HIV) is a lipid enveloped virus. The lipid envelope differs significantly from the lipid membrane of normal human cells: it contains high amounts of cholesterol, that is of importance for the virus-cell interaction (for entry and exit of the virus) at so-called lipid rafts. Cholesterol, as a R-C=C-R compound possesses an oxidazable carbenic bond. The present work suggests the inactivation of HIV by oxidation of viral cholesterol and/or unsaturated fatty acids. For oxidation, the relatively mild oxidant singlet oxygen (1O(2)) might be used. 1O(2) is generated by redoxcyclers (e.g., of the quinone type, such as vitamin K) or by chloramines (e.g., taurine-chloramine). At the 1O(2) concentrations necessary to inactivate lipid enveloped virus in human blood the oxidation-sensible critical hemostasis parameters such as thrombocytes and fibrinogen are only partly inactivated. Therefore, it is proposed to consider generators of 1O(2) as a new form of AIDS therapy.

The physiology and pharmacology of singlet oxygen.

Reactive oxygen species (ROS) are generated by many different cells. Singlet oxygen (1O(2)) and a reaction product of it, excited carbonyls (C=O*), are important ROS. 1O(2) and C=O* are nonradicalic and emit light (one photon/molecule) when returning to ground state oxygen. Especially activated polymorphonuclear neutrophil granulocytes (PMN) produce large amounts of 1O(2). Via activation of the respiratory burst (NADPH oxidase and myeloperoxidase) they synthesize hypochlorite (NaOCl) and chloramines (in particular N-chlorotaurine). Chloramines are selective and stable chemical generators of 1O(2). In the human organism, 1O(2) is both a signal and a weapon with therapeutic potency against very different pathogens, such as microbes, virus, cancer cells and thrombi. Chloramines at blood concentrations between 1 and 2 mmol/L inactivate lipid enveloped virus and chloramines at blood concentrations below 0.5 mmol/L, i.e. at oxidant concentrations that do not affect thrombocytes or hemostasis factors, act antithrombotically by activation of the physiologic PMN mediated fibrinolysis; this thrombolysis is of selective nature, i.e. it does not impair the hemostasis system of the patient allowing the antithrombotic treatment in patients where the current risky thrombolytic treatment is contraindicated. The action of 1O(2) might be compared to the signaling and destroying gunfire of soldiers directed against bandits at night, resulting in an autorecruitment of the physiological inflammatory response. Chloramines (such as the mild and untoxic oxidant chloramine T (N-chloro-p-toluene-sulfonamide)) and their signaling and destroying reaction product 1O(2) might be promising new therapeutic agents against a multitude of up to now refractory diseases.