Coagulation in vitro

Blood coagulation —In vitro platelet aggregation In vitro hemolysis... 

Fig. 4. Influence of 1-ethoxysilatrane on blood coagulation in vitro. Glass start (/), finish (J). Paraffin start (2), finish (4)...

Investigation of coagulation is more easily carried out in vitro. Although the two processes are similar, it is necessary to be cautious in comparing in vivo and in vitro mechanisms. The clot formed in a test tube is different from that formed in the vascular system. Coagulation in vitro consists of a fibrin net in which there exist white and red cells and a small number of platelets. In vivo, the clots consist of large amorphous masses of platelets surrounded by white cells and very few red cells. 

COAGULATION IN VITRO The time in which recalcified plasma will clot after addition of kaolin is termed the activated partial thromboplastin time (aPTT) normal values are from... 

The Hageman factor is a compound of unknown nature that is necessary for blood coagulation in vitro. Some patients lack this factor in the blood, but this does not usually lead to clinical symptoms. It is only responsible for retarded coagulation in vitro. Consequently, the disease is ordinarily discovered as an incidental finding during preoperative preparation of the patient. 

Extrinsic Pathway. Coagulation is initiated when tissue extracts with Hpid—protein properties are released from the membranes of endothehal cells following injury or insult. These substances, collectively designated tissue thromboplastin, complex with circulating Factor VII and in the presence of calcium ions subsequentiy activate Factor X (Fig. 1). In vitro evidence suggests that Factor X can be activated less rapidly through the interaction of kaUikrein [9001-01-8] with Factor VII. 

In Vitro Anticoagulants. A number of substances have been identified that prevent coagulation of the blood when it is removed from the vascular compartment of the body. Most of these substances remove a vital constituent of the blood that is essential in the mediation of transformation of hquid blood into a soHd. 

There is thus obtained bishydroxycoumarin (3). Subsequent pharmacologic and clinical work revealed this compound to be an effective anticoagulant drug in humans. It is of note that none of the synthetic anticoagulants shows in vitro activity. Rather, these compounds owe their effect to inhibition of synthesis by the liver of one of the co-factors necessary for coagulation. 

The procoagulant factors produced by endothelial cells are the coagulation factors von Willebrand factor (WF), F-V, F-VIII, tissue factor (TF), and plasminogen activator inhibitor (PAI), which blocks the activators u-PA and t-PA and counteracts fibrinolysis (G21, FI6). It has been shown that under the influence of complement activation (C9), in response to endotoxin in vitro (C24), in experimental E. coli sepsis in baboons (D30), and after stimulation with TNF (Al, N6), endothelial cells up-regulate the expression of TF, down-regulate TM and inhibit the production of t-PA and PAF. Thus, the balance may shift in the procoagulant direction with a large excess of PAI-1. 

Many of the coagulation factors measured by global coagulation tests have limited stability, and the time and temperature of storage of sample will affect their measurements. Concepts of analyte stability and half-life in plasma extend to markers measured by immunoassay. Markers of platelet activation are affected by artifactual activation in vitro upon collection of the blood specimen. This section will highlight some of the nonanalytical variables that, if uncontrolled, can lead to spurious results and thus affect the interpretation of laboratory data. 

Bagdy D., Barabas E., Bajusz S., Szell E. In vitro inhibition of blood coagulation by tripeptide aldehydes A retrospective screening study focused on the stable D-MePhe-Pro-Atg-H-H2S04. Thromb Haemost 1992 67,325-30. 

Another issue of relevance is that certain biopharmaceuticals (e.g. cytokines such as 1L-1 and TNF Chapter 9) themselves induce a natural pyrogenic response. This rules out use of the rabbit-based assay for detection of exogenous pyrogens in such products. Such difficulties have led to the increased use of an in vitro assay the Limulus ameobocyte lysate (LAL) test. This is based upon endotoxin-stimulated coagulation of amoebocyte lysate obtained from horseshoe crabs. This test is now the most widely used assay for the detection of endotoxins in biopharmaceutical and other pharmaceutical preparations. 

Warfitrin and dicoumaroi prevent coagulation only in vivo and cannot prevent coagulation of blood in vitro (drawn from a patient into a test tube). 

L B. Warfarin does not produce an anticoagulant effect in vitro. It inhibits coagulation of blood only in vivo, because the effect depends upon warfarin s effect in the liver on the production of clotting factors. Warfarin does not require conversion into an active drug. It inhibits the post-ribosomal carboxy-lation of glutamic acid residues in the vitamin K-dependent clotting factors. Therefore, heparin rather than warfarin is used when blood is collected from donors and stored. 

Inhibition of synthesis of prothrombin and coagulation factors Vll, IX, and X Inhibition of platelet aggregation in vitro Activation of plasminogen... 

In contrast to heparin, the coumarinic acid anticoagulants are inactive in vitro like heparin they are active in vivo. The phenylindanedione-type compounds (7) (36) and warfarin (2) produce their in vivo inhibitory effect on the coagulation system by competitively antagonizing the normal activity of... 

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