Clinica Medica I, Universita degh Studi di Firenze

Activation of blood clotting, whose full expression is an increased thrombin generation, can have different aspects and different severity. The clinically most important condition is disseminated intravascutar coagulation (DIC) with a decrease of platelet number, a fall o f plasma concentration of different clotting factors and a reactive h~qgerfibrinolytic condition. A state of disseminated intravascular coagulation can be diagnosed on the basis of a reduced number of platelets, prolonged prothrombin and thrombin times, decreased fibrinogen plasma concentrations and increased levels of fibrin(ogen) degradation products (FDP). These methods, however, are not able to identify states in which a lowgrade thrombin activation occurs. On the other hand, an increased thrombin generation is clinically important, as it often characterizes disorders associated with increased incidence of thromboembolic complications. The aim of this review is to consider different available tests which can be helpful in recognizing a state of increased thrombin generation. BLOOD CLOTTING ACTIVATION

The conversion of prothrombin to thrombin is a crucial event in the activation of blood clotting. In normal conditions, prothrombin activation implies the release of an inactive fragment, Fl+2, fiom the amino terminus o f the prothrombin molecule and the generation of prethrombin 2, which is an intermediate product subsequently splitted to give thrombin. Thrombin is a serine protease and its elective substrate is fibrinogen, but it can act also on other plasma substrates (i.e., factors V, VIII and XIII), and on cell receptors sited on platelets and endothelial ceils. Thrombin, which initially is formed in trace amounts, amplifies the clotting pathway by activating factors V and VIII, that work as accelerating proteins, so furthermore increasing thrombin generKey-words: Coagulation; FibrinopeptideA; Solublefibrinogen complexes; Thrombin; Thrombin-antithrombin complexes. Accepted for publication on July 24, 1990. Res. Clin. Lab. 20, 217-225, 1990.



ation. The first product of the action o f thrombin on fibrinogen is fibrinopeptide A (FPA), thereafter fibrinopeptide B (FPB) is splitted with the formation of fibrin m o n o m e r which polymerizes into fibrin, subsequently cross-linked by factor XIII activated by thrombin itself. Specific receptors for thrombin are present on platelet membrane, so thrombin induces platelet aggregation and initiates the contraction of platelet contractile proteins leading to the release reaction. When thrombin is formed, clotting activation reaction decelerates and thrombin and other coagulation activated factors such as factors V, VIII, IX and X are inactivated. Thrombin is mainly inactivated by antithrombin III by forming a thrombin-antithrombin (TAT) complex. The inactivation of thrombin (and factor Xa) needs heparin or heparan sulfate as accelerating cofactors. Moreover, thrombin in the presence o f an endothelial cofactor (thrombomodulin) and calcium ions activates also protein C by cleaving a b o n d at the aminoterminal end of the heavy chain of the protein, and so releasing a small dodecapeptide whose molecular weight approximates 1,400 daltons. The major thrombin antagonist is represented by the plasma protein antithrombin III (ATIII), which forms with thrombin a stable inactive enzymeinhibitor complex. The direct measurement of thrombin in plasma is not feasible, due both to the paucity of circulating thrombin rapidly inactivated by ATIII and to the absence of reliable methods to directly determine the enzyme activity in plasma. Thus, the thrombin generation rate can only be measured indirectly by the products specifically released during conversion of prothrombin to thrombin (Fl+2 fragment) or by thrombin activity on fibrinogen. No information can be obtained from thrombin activity on platelets. The decrease in platelet counts and the presence of exhausted circulating platelets may indicate the intravascular effects of thrombin, but this is not a specific finding because it can be induced by other aggregating stimuli. Similarly, no exact information can be obtained from thrombin activity on factors V and VIII. During blood clotting the large amount of thrombin which is produced destroys both factor V and factor VIII activities, whereas factor VIII-related antigen is unaffected by thrombin. O n the contrary, thrombin at low concentrations enhances factor VIII activity 3°. Thus, it was thought that the factor VIII-related antigen/factor VIII biological activity ratio would be an index of clotting activation 15. Actually, an increased ratio should be considered as an index of DIC, while a decreased ratio may represent an index of lowgrade clotting activation 34. However, the opposite effects of different thrombin concentrations on factor V and factor VIII activities and possible effects of other enzymes (such as plasmin) make this approach o f difficult interpretation in conditions o f low-grade thrombin generation. In the last years there has been increasing interest in the study of some peptides which can be considered specific markers o f an increased formation o f thrombin in blood 8. Different methods for the detection of fibrinogen products by thrombin action were proposed, but many were a b a n d o n e d because o f their p o o r sensitivity and/or specificity.

Soluble fibrin-fibrinogen complexes assays Soluble intermediate products o f fibrinogen are produced during the conversion of fibrinogen to fibrin H,4s, and they are a group of substances with 218


different molecular weights and differing by several characteristics. When thrombin acts on fibrinogen and splits FPA and FPB, fibrin m o n o m e r is formed. If fibrin monomer reaches a critical concentration (5% of all fibrinogen), it polymerizes into fibrin gel. Otherwise it forms complexes either with fibrinogen or fibrin(ogen) degradation products or with still soluble fibrin oligomers 46. These high molecular weight complexes are initially soluble. The presence of thrombin-induced fibrinogen derivatives in the blood was considered to be a sign of hypercoagulability 2' ~9.20,45. They were presumed to be formed in various disease states, particularly DIC 19,56, and several assays were proposed to detect and quantitate these complexes in clinical blood samples, i.e., agarose gel exclusion chromatography, Sepharose-fibrinogen affinity chromatography, paracoagulation assays (protamine sulfate test and ethanol gelation test), agglutination tests employing erythrocytes complexed with fibrin monomers and photometric tests based upon the capacity of fibrin monomers to accelerate the activation of plasminogen to plasmin by tissue plasminogen activator. Most of these methods are nowadays of historical interest. Agarose gel chromatography for the identification of complexes with higher molecular weight than native fibrinogen has been applied to clinical blood samples by FLETCHERet al. 2, 19. Affinity methods are based upon immobilization of insoluble derivatives of human fibrinogen to CNBr-activated Sepharose 6B 2~,3~. When plasma was passed through columns containing immobilized fibrinogen, the fibrin of the sample was absorbed and it could be subsequently eluted with buffers 2~,44. Even if the analysis of these complexes by suitable methods enabled to recognize that they are mainly represented by fibrinogen derivatives and that therefore they are to be considered as an evidence of blood clotting activation, chromatographic methods have not b e e n widely applied to clinical plasma samples because they are time consuming, not suitable for the analysis of a large number of samples and require instrumentation not easily available in routine laboratories. Moreover, the diagnosis would be made too late. Paracoagulation tests are based upon the property of fibrinogen and of some of its derivatives to precipitate by varying the medium 16 either through the addition o f organic solvents like ethanol 2° or the addition of charged substances such as protamine sulfate 4a. Unstable and easily precipitating complexes are formed between fibrinogen, soluble fibrin and various degradation products. Independently of differences among different paracoagulation assays employed in the past, it should be stressed that they are no more to be recommended because they are only qualitative assays and have very limited specificity and sensitivity 56. A semiquantitative assay for the detection o f soluble fibrin in plasma is represented by the agglutination reaction of glutaraldehyde-treated erythrocytes coated with purified fibrin monomers 26. The test is based on the property of circulating fibrin monomers to aggregate with monomers present on erythrocytes. Positive tests were reported in DIC and in conditions with marked clotting activation, while no evidence exists for their usefulness to detect states o f low-grade thrombin generation 48. A more recent test to determine the concentrations of fibrin monomers is based on their capacity to enhance the activation rate of plasminogen to plasmin by tissue plasminogen activator 57. The amount of circulating fibrin mono219


mers is determined by assessing the enhancement of plasmin formation through measurement of its amidolytic activity using a chromogenic substrate. This test seems to be reproducible and it has b e e n recommended for the screening o f DIC, although some doubts exist on its capacity to detect lowgrade thrombin generation 57 and studies relating to the critical evaluation of these more recent assays are not available.

Fibrinopeptide A assay FPA is a peptide with molecular weight of t,500 daltons composed of 16 amino acids, splitted from the aminoterminal portion of the two A chains of fibrinogen by thrombin, which acts on bonds not cleaved by plasmin ~6. Thus, FPA plasma concentration is a specific index of thrombin action. Since FPA half-life is very short (3-5 rain), its plasma concentration gives reliable information on the actual blood clotting activation 3~,37. A delicate task for obtaining a reliable determination of FPA levels is represented by blood sampling and subsequent manipulation of samples. I n fact, during sampling injured skin and vein tissues can release tissue thromboplastin, and prolonged venous stasis can cause clotting activation. Moreover, thrombin can be formed in trace amounts before b l o o d and anticoagulant solution are completely mixed. Thus, blood must be carefully withdrawn by trauma-free venipuncture with rapid blood flow without venous occlusion by means of needles of adequate lumen (19-gauge) and by the two-syringe technique. T h e first 3 or 4 ml are discarded and then blood is collected into an iced polypropylene syringe containing anticoagulant solution. Another critical step before FPA assay is represented by fibrinogen elimination from the sample. The complete removal of fibrinogen is particularly critical because commercial antibodies do not completely discriminate free FPA from FPA still present in the fibrinogen molecule. Fibrinogen can be removed by different physical methods such as precipitation, dialysis and absorption. The most commonly employed method is absorption by bentonite 25. Relatively high bentonite concentrations (20 mg/ml plasma) are requested because low concentrations are not only unable to completely remove fibrinogen, but cause the activation both of coagulation and fibrinolysis. The preparation of bentonite solution needs particular care and solution must remain under continuous stirring. Some differences exist among various commercial bentonite preparations, and a preliminary evaluation o f bentonite concentration to be employed is necessary for each batch. Another factor which can significantly affect the FPA assay is the phosphate content of FPA, which causes changes in the affinity for the different antibodies employed. About 30% of FPA contains a phosphate groupl° covalently attached to the third amino acid, serine, and was designated phosphorylatedFPA (FPA-P). Experimental studies 29 indicate that differences in FPA concentrations measured with different antisera could be accounted for by their different reactivity with FPA-E It is to be stressed that in patients with elevated FPA levels the mean FPA-P content o f fibrinogen is significantly higher than in subjects with normal FPA levels. Therefore, blood clotting activation may lead to a high phosphate content both of fibrinogen and free fibrinopeptide A in plasma 29. 220


Many different techniques have been employed to measure FPA, such as electrophoretic or chromatographic methods, which are now only of historical interestS2, ~2,45. Nowadays, very sensitive radioimmunoassay~3,36 and enzyme immunoassay 49 are commercially available to measure FPA concentration. In many different groups of patients with thrombotic diseases or with conditions at thrombotic risk elevated plasma levels of fibrinopeptide A were rep o r t e d 7' ~ ' 40' 42' 52' 53" 58. Of particular relevance is considered FPA in cardiologyt7, 18,24,33,54. FPA has proved to be a useful clue that has advanced our understanding of different coronary syndromes and provided convincing evidence of the existence of a clear clotting activation state in most of acute myocardial ischemic syndromes, leading to the widespread use of blood clotting inhibiting treatment. It is to be stressed that FPA cannot be employed to make a clinical diagnosis, but only to prove the existence of an increased thrombin generation. Since technical factors involved in blood sampling may lead to falsely elevated plasma FPA levels ~5, some authors have proposed to use spot urinary FPA measurement which correlates with a simultaneously obtained plasma level 1,55. However, this approach does not permit to evaluate the actual thrombin generation because the urinary concentration of FPA can result either from a short-lasting high-grade clotting activation followed by rapid normalization or from a continuous low-grade activation. Thus, plasma FPA measurement seems to be the best tool to study thrombin generation.

Fl+2 fragment assay Another assay which permits to study a condition of clotting activation is that o f Fl+2 fragment. During prothrombin activation inactive F1+2 fragment is released. Therefore, F1+2 fragment assay measures the prothrombin cleavage rate by factor Xa rather than thrombin generation and activity. Thus, this assay is not comparable with FPA assay. Both radioimmunoassays ~7,5~ and enzyme immunoassays 3g.47 were set up for the measurement of Fl+2 fragment. Some data have been published in the last years by the group of BAUER and ROSENBERO4-9, who studied the relationships among the concentrations of several clotting activation-related peptides. A dissociation between factor Xa activity (assessed by F1+2 fragment) and thrombin generation (assessed by FPA) was observed in particular conditions, e.g. in asymptomatic individuals with hereditary antithrombin III 5 or protein C 4 deficiencies. Moreover, no direct correlations could be established between Fl+2 and either FPA or protein C peptide measurements 9. This fact may result in part from the relatively prolonged survival of Fl+2 in human circulation as compared with FPA 37 or protein C peptide 6. However, a more rapid in vivo neutralization rate for thrombin than for factor Xa could also play a role 5. A highly significant positive correlation between aging and plasma levels of Fl+2 was reported 9. Moreover, Fl+2 assay has been reported to be an accurate marker of factor Xa activity on prothrombin in the blood of individuals treated with oral anticoagulants 14. Protein C activation peptide assay H u m a n protein C consists of a hea W chain and a light chain that are linked by a single disulfide bridge. During the activation of protein C by thrombin b o u n d to thrombomodulin on the surface of vascular endothelium a 221


bond is cleaved at the aminoterminal end of the heavy chain of protein C, so releasing a protein C activation peptide. This peptide can be measured by a radioimmunoassay prepared by BAUER et al. 6. Significant elevations with aging were reported, but their size was smaller than for F1+2 9. No data from other groups were reported and routine methods are not available for clinical use. Thus, at present the role of this approach has not been fully elucidated.

Thrombin-antithrombin complexes assay Thrombin can combine with its major antagonist ATIII causing the formation of a stable inactive enzyme-inhibitor complex. A sensitive and specific radioimmunoassay able to quantitate the levels of TAT was firstly reported by TEITEL et al. 51. Enzyme immunoassays were also reported 38 and are now commercially available for routine laboratory. Preanalysis bias sources seem to be less critical for TAT determination than for FPA assay. However, the two tests give different information because • they are related to different steps. TAT complexes indicate thrombin bound to ATIII. In theory, if all thrombin was combined with ATIII no FPA would be formed. FPA assay gives a specific measure of actual thrombin action and only active (not inactivated) thrombin plays an important role. Thus, the two assays are not alternative but complementary. Increased plasma TAT levels have been reported in disseminated intravascular coagulation and in various disease states characterized by activation o f the clotting system with marked thrombin generation 3,13.2~,28,38,41,4z,50,51. However, the sensitivity of TAT assay in detecting states characterized by a low-grade thrombin activation still remains to be elucidated. A comparison among the assays of FPA, Fl+2 fragment and TAT has been reported in patients with acute leukemia with increased thrombin generation 7. These results indicate a lower sensitivity of TAT in comparison with the other tests for detecting clotting activation. COMMENT

Several methods have been proposed to study thrombin generation, but some were never used for clinical purposes due to their difficulty, and some others were abandoned for their poor sensitivity a n d / o r specificity. Recently, several new tests became available, but their usefulness has not yet been fully elucidated; TAT assay seems to be promising, but its sensitivity seems relatively poor and it needs further validation. For these reasons and for the bulk of data in different clinical states FPA measurement with careful standardization of pre-test conditions is to be considered at present as the 'gold standard' among laboratory methods which identify an increased thrombin generation.

SUMMARY Blood clotting activation causes an increased t h r o m b i n generation. T h e direct m e a s u r e m e n t of plasma t h r o m b i n is not feasible due to the absence of reliable methods. Thus, only indirect approaches are possible. Different markers of t h r o m b i n generation have b e e n proposed in the past years. Some methods are nowadays of historical interest. A m o n g various methods reported the 'gold standard' is fibrinopeptide A assay, while t h r o m b i n - a n t i t h r o m b i n complexes assay needs further validation to be employed as a reliable index of increased t h r o m b i n generation, a n d more recent methods (such as those for F l + 2 fragment a n d for protein C activation peptide) are not available for routine use.


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Markers of increased thrombin generation.

Blood clotting activation causes an increased thrombin generation. The direct measurement of plasma thrombin is not feasible due to the absence of rel...
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