Primary hemostasis

Primary hemostasis is maintained by the action of platelets, which interact with the damaged vessel wall, and with each other, to form a platelet plug, in collaboration with coagulation factors. Tests assessing platelet function determine either overall platelet reactivity (bleeding time) or specific functions such as adhesion, aggregation, or release. Most of these latter tests are used to screen patients suspected of having a specific hemostatic defect.

Simple devices for measuring bleeding time are commercially available. However, there are only a few indications for and considerable limitations on their use. Plasma coagulation

Plasma coagulation, the second part of the hemostatic defense mechanism, involves a series of linked coagulation protease-zymogen reactions, ultimately resulting in the formation of fibrin molecules (CDFigureJ). In the current hypothetical model of coagulation, thrombin generation is induced by the assembly of the tissue factor-factor Vila complex, previously identified as the starting point of the extrinsic route of coagulation ( D&vi&etal 1991). Tissue factor is a glycoprotein which is not normally present in the circulation in detectable quantities, but can be induced in monocytes or endothelial cells by a variety of stimuli such as cytokines. Factor VII is bound upon exposure of tissue factor at the cell surface, and after its activation, the complex catalyzes the activation of factors IX and X, resulting in a rapid onset of factor X activation and subsequent conversion of prothrombin to thrombin. Thrombin is a key enzyme in the clotting cascade, enhancing its formation rate by several feedback loops. Thrombin eventually cleaves fibrinogen, yielding fibrin molecules which may polymerize via the action of activated factor XIII to form insoluble fibrin, and vascular occlusion may occur.

CD Figure 1. Model of the hypothetical coagulation cascade. Products of coagulation activation are shown in boxes: TF-Vlla, tissue factor-factor Vila complex; IXp, factor IX activation peptide; Xp, factor X activation peptide; F1+2, prothrombin fragment 1+2; FPA, fibrinopeptide A; D-dimer, fibrin split product; TAT, thrombin-antithrombin complex. The following natural inhibitors are shown: TFPI, tissue factor pathway inhibitor; AT, antithrombin; PCa, activated protein C (which cleaves activated factors V and VIII); TAFI, the recently identified "thrombin activatable fibrinolytic inhibitor". Coagulation proteins are given in roman numbers; PK, prekallikrein; HK, high-molecular-weight kininogen.

Assessment of the functional integrity of the coagulation cascade is classically performed with clotting assays. The activated partial thromboplastin time determines the classical contact activation pathway by activating factor XII, followed by conversion of factors XI, IX, VIII, X, and V, and prothrombin. Clotting is started by adding an activator of the contact route and calcium to citrated plasma, and the clotting time is recorded. The assay is suitable for detecting either functional (inhibitors) or absolute deficiencies in any of the intrinsic route factors, or for measuring the effects of anticoagulant treatment, particularly heparin.

The classical extrinsic route, i.e. the tissue factor-factor VII pathway activates factor X, prothrombin, and fibrinogen, and is monitored by the prothrombin time. In this assay clotting is started by adding thromboplastin (a tissue-factor-containing preparation) and calcium to citrated plasma, and recording the clotting time. This assay is sensitive to functional abnormalities of primarily vitamin-K-dependent clotting factors (factors VII and X, prothrombin), and measures functional or absolute deficiencies of any of these clotting proteins. Furthermore, the test is particularly sensitive to the effects of oral anticoagulant agents (coumarins).

The thrombin (clotting) time specifically measures the final conversion of fibrinogen to fibrin, induced by adding thrombin and calcium to citrated plasma, and recording the clotting time. It is sensitive to reduced levels of fibrinogen or to abnormal clotting activity of this molecule.

By employing a combination of one or more of these clotting assays, a general indication can be obtained of the presence of any abnormality in the clotting mechanism or any effect induced by anticoagulant treatment.

In addition to these clotting assays, more sensitive and specific assays have been developed during the past decade. These assays are aimed at detecting products of the actual clotting process, i.e. activation peptide fragments, which are generated upon conversion of some of the clotting zymogens, free enzymes, or enzyme-inhibitor complexes (,B.a.ye.L.┬žn.d We.!iZ...1994). By developing specific antibodies against these reaction products of coagulation (and fibrinolysis), immunoassays were developed for quantitating the plasma levels of the activation markers. This methodology provided a markedly increased level of detection of coagulation activation compared with the less sensitive clotting assays, and showed that even normal individuals had low basal levels of coagulation activity. This enabled coagulation derangements in disease processes to be studied at a very early stage.

Assays for measuring activation fragments, released upon activation of coagulation zymogens such as prothrombin fragment F1+2, fibrin monomers, and fibrinopeptide A, or enzyme inhibitor complexes (thrombin-antithrombin) are commercially available, and others are being developed for clinical use. Most are enzyme-linked immunosorbent assays (ELISA) and radio-immunoassays, and are laborious and expensive to perform. Therefore most of these assays are still unsuitable for routine clinical application.

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