C.R. Spillert

Effect of endotoxin and silver ion on the clotting time of blood

Endotoxin (Lipopolysaccharide, LPS) is a part of the outer membrane of the cell wall of gram-negative bacteria. LPS induces activation of the coagulation cascade in humans. This procoagulant effect on citrated whole blood produces a shortened clotting time. Silver ion (Ag/sup +/) denatures the anticoagulant proteins and affects the intrinsic pathway of blood coagulation by producing a shortened clotting time. This in vitro study was performed by mixing human citrated whole blood (CWB) with LPS, silver nitrate (AgNO/sub 3/), and the combination of the two. The clotting time, rate of clot formation, and the maximum strength of the clot were determined. The results show that the combination of LPS and Ag/sup +/ significantly reduces the clotting time (p<.002).

In vitro investigation of streptokinase activity using Sonoclot coagulation analysis

Streptokinase (SK) therapy has become a common clinical intervention in the treatment of acute coronary syndromes. Recent clinical trials indicate that adjuvant treatment with low molecular weight heparin (LMWH) or bivalirudin (BV) may attenuate fibrinolytic-induced procoagulant activity. We investigated the effects of SK with LMWH and BV on the clotting of whole blood using a Sonoclot/sup /spl reg// Coagulation Analyzer. Human citrated whole blood was spiked with SK and LMWH at final concentrations of control, 5 U/mL SK, 0.2 U/mL LMWH, and 5 U/mL SK:0.2 U/mL LMWH. The samples (n=10) were incubated at 37/spl deg/C for 10 minutes and recalcified with CaCl/sub 2/ to initiate clotting. Tracings were obtained using the Sonoclot Signature Viewer/spl trade/ software, and the clotting time (CT) and clot viscosity were noted. The experiment was repeated (n=8) using 5 /spl mu/g/mL BV in place of LMWH. The Sonoclot tracings provide a whole-picture analysis of the effects of SK and adjuvant anticoagulants on haemostatic and fibrinolytic mechanisms. Implementation of coagulation monitoring protocols for SK therapy may aid in clinical diagnosis.

A functional clotting assay to monitor low molecular weight heparin dosage

Low molecular weight heparin (LMWH), an anticoagulant drug, has seen increased use in a variety of clinical situations. Mercuric ion (Hg/sup 2+/) has been shown to increase procoagulant activity via Tissue Factor (TF) stimulated production of Factor Xa, a pathway that has been linked to many thrombotic disorders. The effects of Hg/sup 2+/ on heparinized blood may provide valuable insight into the effectiveness of LMWH to counteract TF-induced hypercoagulability. We performed in vitro studies by spiking human citrated whole blood (CWB) with HgCl/sub 2/ and LMWH at final concentrations of 0.01 % HgCl/sub 2/, 0.5 U/mL LMWH, and a combination of these agents. The samples (n=19) were incubated at 37/spl deg/ for 10 minutes and recalcified with 0.1 M CaCl/sub 2/ to initiate clotting. The clotting or recalcification time (RT) was measured with a Sonoclot Coagulation Analyzer. The results indicate that HgC12 was able to significantly (p<0.0001) reduce the anticoagulant ability of LMWH. The data warrants a reassessment of current LMWH monitoring and therapeutic protocols.

Effects of Protamine Sulfate, Heparin and Mercuric Chloride on Erythrocyte Sedimentation Rates

Protamine Sulfate (PS) is commonly administered in cardiovascular surgery to neutralize the anticoagulant effect of low-molecular-weight heparin (LMHW). The purpose of this study was to find the individual and combined effects of PS, LMWH, and mercuric chloride on the Erythrocyte Sedimentation Rate (ESR) of human blood. Our results showed that the samples (n=11) containing mercuric chloride retarded ESR values compared to control values. Samples (n=11) containing mercuric chloride coupled with either PS or LMWH showed a synergistic effect and retarded ESR values furthermore. Finally, samples (n=11) containing all three reagents did not show a significant difference in ESR values when compared to the control.

Effect of tissue factor on plasma and packed RBC clotting parameters

Tissue factor (TF) is a transmembrane glycoprotein and the main triggering element for blood coagulation. Expression of TF on the cell surfaces and its appearance as a soluble molecule are characteristic features of acute and chronic inflammation in conditions such as arteriosclerosis leading to stroke and myocardial infection. A study was performed on human citrated whole blood, by separating plasma from the red blood cells. Ten microlitres of 25% tissue factor (TF) was added to both the plasma and packed red blood cells (RBCs), and the clotting time, rate of clot formation and the maximum strength of the clot determined. TF bearing samples clotted more rapidly and had greater clot strength when compared to that of control aliquots.

The effect of copper ion on blood coagulation

The effect of copper ions on the blood coagulation time was determined. In the experiment that was performed in vitro, there were three different aliquots for each blood sample. The three aliquots were composed of the control, 0.05 mM CuSO/sub 4/, and 0.1 mM CuSO/sub 4/. Thirty two microliters of 0.1 M CaCl/sub 2/ was then added to each aliquot to initiate blood coagulation. When the copper ion concentration was increased, there was a significant increase in blood coagulation time.

In vitro investigation of the procoagulant of silver ion

Silver ion, a potent antimicrobial and procoagulant, is hypothesized to achieve its haemostatic effect by binding anticoagulant proteins. We performed in vitro studies to gain more insight into its mechanism of action. 10 /spl mu/L of 5% AgNO/sub 3/ was added to 1 mL human plasma (n = 4), forming a precipitate. The precipitate was recovered through centrifugation and iteratively washed with saline. The supernatant(s) and precipitate were mixed with Coomassie blue stain and protein content was measured via spectrophotometric absorbance. An analysis of variance indicated significant (p<0.0001) differences between all groups. Multiple comparison tests showed a significant (p<0.001) increase in absorbance from the final wash and dye blank to the precipitate, indicating the presence of proteins in the precipitate. Silver ion may be precipitating anticoagulant proteins as its mechanism of action.

The effect of copper ions on sedimentation rates of erythrocytes

Whether copper sulfate affects the sedimentation rate of erythrocytes in blood was investigated. We performed the experiment by placing by 1ml of blood in plastic vials. After that, we added 10/spl mu/l of 1% CuSO/sub 4/ solution to an aliquot. Then, we put 10/spl mu/l of 5% of CuSO/sub 4/ to 1ml of the same blood into different vial. Following this procedure, we incubated the blood with the various concentrations of CuSO/sub 4/ to 37 degrees Celcius for ten minutes. Then, we determined the sedimentation rates of the red blood cells (RBCs) for the control, the blood that contained .001% of CuSO/sub 4/, and the blood that contained .005% of CuSO/sub 4/. We repeated this experiment with fourteen (n=14) different samples of blood. The results indicate that there was a statistical significance (P<.05) in the sedimentation rate of the blood that contained the .005% of CuSO/sub 4/ and the control were different In addition, there was a statistical significance (P<.05) that there was a difference in sedimentation rate between the .001% CuSO/sub 4/ and .005% CuSO/sub 4/. The results also show that there is no difference in sedimentation between .001% and the control.

Contact with glass interferes with the Erythrocyte Sedimentation Rate (ESR)

The sedimentation rate (sed rate), also called, the Erythrocyte Sedimentation Rate (ESR) is a blood test for inflammation in the body. If it is elevated, then there is an inflammatory process in the body which can be monitored. The test is broad and non-specific; however, it may determine the effectiveness of therapy for the inflammatory condition. We performed studies on human citrated whole blood by adding 100 microlitres of 1% of MethylCellulose to each of glass and plastic tubes with the blood. The samples (n=12) were incubated at 37/spl deg/ for 10 minutes. Later, the blood from each of the tubes were placed in pipettes, sealed and left undisturbed for an hour. The ESR was measured, and it was found that the sedimentation rate of the blood in plastic settled at a higher rate than the one in glass.

Combined effect of mercuric ion and silver ion on the clotting time of blood

The mercuric ion (Hg/sup 2+/) has been shown to have a procoagulant effect on citrated whole blood through its excitation of the extrinsic pathway. The silver ion (Ag/sup +/) affects the intrinsic pathway of blood coagulation also producing a shortened clotting time. This in vitro study involved mixing citrated whole blood (CWB) with mercuric nitrate Hg(NO/sub 3/)/sub 2/, silver nitrate AgNO/sub 3/, and the combination of the two. The eight samples were incubated for ten minutes at 37/spl deg/C and were analyzed by an Amelung KC 4A clot detector. The results show that the combination of Ag/sup +/ and Hg/sup 2+/ significantly reduces the clotting time (p<.002).