What is truly remarkable about this system is that we neither bleed to death nor clot solid within the first few hours of life.
The classic coagulation cascade was introduced by Biggs and MacFarlane in the early part of 1964. It is unlikely that they had any idea of the impact their hypothesis would have on the investigation and understanding of hemostasis over the next 30 years.
According to their cascade theory the coagulation mechanism is composed of an extrinsic and intrinsic pathway. The extrinsic pathway begins with the interaction of the phospholipid tissue factor with factor VII to activate factor X. Activated factor X is also generated by the intrinsic pathway, which is initiated by contact activation of factor XII (Hageman factor) when it comes in contact with the negatively charged surfaces underlying the endothelium.
This beautifully simple explanation of the complex interaction of the coagulation factors was immediately accepted world wide. It was assumed that the extrinsic system was the minor pathway simply because there were so few factors involved and because it was soon shown that birds and reptiles, species which are obviously lower on the evolutionary scale, posses only an extrinsic system. The intrinsic system was by far the most important because it is more complex, present only in higher life forms, and the two most common inherited bleeding abnormalities are in this pathway.
Even though the rest of the "coagulation gurus" were running with this explanation, Rosemary Biggs recognized that all was not well in the world of "cascade coagulation". How is it that patients deficient in the contact factors, for example factor XII, did not have a bleeding problem. John Hageman, the first patient identified with factor XII deficiency, would die years later not of bleeding complication but rather from a pulmonary embolus. Hageman's chief problem all his life was not bleeding problems but recurrent infections. As well, if in fact there are two systems, even with the extrinsic being the lesser of the two, why do hemophiliacs bleed? She went back and repeated an experiment she had originally published in 1951 in which she discovered that when you perform a prothrombin time on factor VIII or IX deficient plasma using a physiologic concentration of tissue thromboplastin, the result was abnormal. Rosemary published these findings in a second paper at the end of 1964 and suggested that the factor VII/Ca++/tissue factor complex was of much more significance than the cascade hypothesis suggested. This paper was almost completely ignored.
Towards the end of the 80's and into the early 90's, studies began to suggest that indeed Biggs had been correct all along and there is in fact no separation of the two pathways. Tissue factor and factor VII are the central controlling key in the entire hemostatic mechanism. In fact, the initial steps in the intrinsic system are important to us in our coagulation tests but invivo are of major significance for there involvement in the inflammatory mechanism.
Current concepts indicate that at the site of vessel injury, factor VII binds to tissue factor and is immediately activated from its zymogen to its enzymatically active form. This results in what can be called a "two-unit enzyme". The activated factor VII being the catalytic component and the tissue factor being the regulatory or rate controlling part. This tissue factor/factor VIIa complex binds and activates factor X and factor IX. During the activation of factor X a small piece of factor X is split off and washed away in the plasma. We will return to this factor X activation peptide in a few minutes. The activated factor X, in the presence of factor V, Ca++ and the platelet membrane, converts prothrombin to thrombin, which in turn converts fibrinogen to fibrin. As well the factor VII/tissue factor complex also activates factor IX. The activated factor IX, in the presence of factor VIII, Ca++, and the platelet membrane, activates more factor X which generates more thrombin. Some of this thrombin activates factor XI which then accelerates the activation of factor IX. The net result is an amplification process with the ultimate, localized generation of large amounts of thrombin and the explosive conversion of fibrinogen to the fibrin clot.
To review then, it is obvious that the division of the coagulation mechanism into the intrinsic and extrinsic system, albeit convenient, is not justified. In fact we have returned to an understanding of coagulation which was originally introduced by Morawitz in 1905. 90 years to come full cycle. Once again we are recognizing the critical importance of factor VII and tissue factor. This really shouldn't be too much of a surprise. After all, when investigators tried to identify some coagulation link to MI the only direct association existed between factor VII levels and, to a lesser extent, fibrinogen levels.
During the late 70's and early 80's the components of the natural anticoagulant system were identified. This system was thought to consist of antithrombin III (ATIII), and the protein C and S complex.
Antithrombin III is produced in the liver and circulates free in the plasma. It is attracted to the negatively charged heparin or heparin proteoglycan on the surface of endothelial cells. Once bound a configurational change occurs such that it is now able to bind thrombin and the other serine proteases (XI, IX, X) and inactivate them.
The anticoagulant proteins C and S are produced in the liver and are vitamin K dependant. If thrombin is produced in excess of what can be neutralized by ATIII, the thrombin binds to thrombomodulin on the surface of the endothelial cell. This exposes the Ca++ receptor site on the thrombomodulin and Protein C binds and is activated. The activated Protein C binds to Protein S, already bound to its specific receptor on the surface of endothelial cells. The Protein C&S complex is then able to destroy activated factors V and VIII. For the last 15 years these three anticoagulants; ATIII, Protein C, and Protein S, have been touted as the factors essential for the normal balance of the hemostatic mechanism. If this were true, why is it impossible to identify an abnormality in any of these factors in the vast majority of patients with thromboembolic disease?
A very significant development occured in the Protein C and S system in 1993. A Swedish group identified what they at first thought was a new cofactor in the Protein C&S system. The idea was that there is a cofactor which is essential if activated Protein C is to bind to Protein S. This isn't quite the way things turned out. It is now known that the defect lies in factor V. They have been able to show that approximately 50% of all patients with thrombotic disease have the factor V:Leiden, ie, a genetic mutation at position 506 in factor V where argenine has been replaced with glutamine. This substitution is itself due to the fact that the gene controlling the production of factor V has itself under gone a single nucleotide change. guanine at position 1691 has been substituted for adenine. Factor V Leiden has also been called "aPC-resistance" factor and a modified APTT procedure, called the "COATEST APC resistance" test, has been developed. This test is available for evaluation from Chromagenix. The substitution eliminates the site at which activated Protein C cleaves factor V. As a result factor V is not inactivated and continues to contribute to the coagulation cascade leading to thromboembolic disorders. As an interesting side light, recent studies have shown that women who develop thrombosis while on the pill are 8 times more likely to have this inherited factor V defect and that this abnormality is at least 10 times more common than all other known genetic risk factors for thrombosis; protein C, protein S and antithrombin III deficiency, together.
The third newly rediscovered anticoagulant is Tissue Factor Pathway Inhibitor (TFPI). TFPI was first described almost 50 years ago and as a result has been known by a variety of names including;
In 1991 the International Society on Hemostasis and Thrombosis decided to name this anticoagulant "Tissue Factor Pathway Inhibitor" (TFPI). TFPI is produced by endothelial cells and monocytes and inhibits activated factor X and the factor VIIa/tissue factor complex. TFPI is a 276 amino acid enzyme which is unique in that it has 3 functional sites or Kunitz domains. Due to the twisted shape of these functional sites they are sometimes called Kringle sites because they look like the twisted breads made in New York. The first Kunitz inhibits the tissue factor/VIIa complex, The second Kunitz inhibits activated factor X, and the third Kunitz may be the TFPI binding site to lipoproteins. TFPI exists in three compartments or pools. Most of the TFPI is bound to the endothelial cell surface. Of the remaining TFPI, 10% is bound to the platelet membrane and 90% is transported in the lipoprotein fraction of the plasma. TFPI is a heparin cofactor and is released from endothelial cells by heparin administration. It is a more potent antithrombotic than heparin or ATIII. It is able to affect both the intrinsic and extrinsic system and does not compromise platelet function.
In 1856, Virchow pointed out that thrombosis occurs as a result of;
These are often referred to as Virchow's triad. We now realise that the blood vessel wall plays the controlling role in the stimulation of coagualtion and the prevention of coagualtion. The inner surface of the blood vessel is responsible preventing coagulation while the middle and outer layers stimulate coagulation.
It doesn't take a rocket scientist to figure out that a lot more patients die of thrombosis, that is, a hypercoaguable state, than die of hemorrhage, that is, a hypocoaguable state.
Hypercoaguable states can be either inherited or acquired.
Inherited deficiency of any one of the natural anticoagulants will result in thrombosis. American studies show that less than 25% of all patients with thrombotic disease have a deficiency in ATIII, Protein C or S. Scandinavian studies say less than 8% are deficient in these factors. If we average these results it means that approximately 85% of thrombotic episodes occur because a patient has some other inherited defect (aPC or TFPI) or some acquired defect. As already mentioned approximately 50% of patients studied have an aPC deficiency.
Acquired hypercoaguable states include;
Antiphospholipid antibodies, also called lupus anticoagulants are, according to some authors, the most common cause of an acquired thrombotic disease. A great deal of work has gone into trying to identify the exact nature of the lupus anticoagulant and how it works. An interesting possibility is that the lupus anticoagulant has specificity or cross-reactivity for TFPI. Once the APA/TFPI complex forms the coagulation mechanism is not inhibited and clot forms. Several american investigators have suggested that all patients with thrombotic disease must be investigated for the presence of the lupus anticoagulant. Bick suggests that one of the lupus anticoagulant tests, such as the DRVVT as well as IgG and IgM anticardiolipin levels on two occasions at least 12 weeks apart should be performed.
Recent studies have shown that the major binder of TFPI is LDL with lesser amounts bound by HDL and VLDL. What has not been determined to this point is wether bound TFPI is in fact available as an anticoagulant. If bound TFPI is physiologically unavailable this could well explain the relationship between liporproteins and atherosclerosis. The more LDL the more bound TFPI and the less inhibition of the coagulation mechanism.
Until recently the laboratory involvement in thrombotic disease has been the monitoring of heparin and warfarin therapy. This is definitely closing the barn door after the horse has been stolen. It makes much more sense to try to identify those patients who are predisposed to thrombosis.
Tests are currently available for Protein C, Protein S and ATIII, but are probably not cost effective due to the fact that less than 15% of all thrombotic patients have deficiencies in these factors.
It seems only logical that tests for antiphospholipid antibodies, activated protein C resistance and TFPI should be performed in large enough numbers to determine wether or not any or all of these defects are the cause of thrombotic disease.
Antiphospholipid antibody tests:
Simplified DVVtests are available which can be run manually or fully
automated.
APC resistance tests:
A kit procedure called the "COATEST APC resistance" test, is
available from Chromagenix or American Diagnostica. The procedure is
a modified activated PTT. However, it requires special handling of
fresh patient plasma and it can not be run on patients receiving
warfarin or patients with antiphospholipid antibodies. A new test has
been described in the April 1995 issue of Blood which appears to be
more sensitive yet much easier to perform. The test plasma is diluted
and then duplicate samples are incubated in factor V deficient plasma
and tissue factor. After incubation calcium is added to one duplicate
and calcium and APC is added to the other. The clotting times are
recorded. Both clotting times will be the same in the presence of
Factor V Leiden.
As an aside it is important to note that activated protein C
resistance interferes with functional Protein S assays. All patients
which have been previously diagnosed as having functional Protein S
deficiency should be re-investigated for APC resistance.
TFPI tests:
Both functional and antigenic tests are available for TFPI. There are
two functional assays.
1. The Sandset assay is a time consuming, complex, chromogenic
substrate assay in which diluted plasma is incubated with tissue
factor, activated factor VII, and factor X. The amount of activated
factor X generated is inversely proportional to the amount of TFPI
present.
2. The modified PT is not as complex but requires the use of anti-
TFPI antibodies. A prothrombin time is performed on plasma with and
without added anti-TFPI antibodies. The difference in times is
proportional to the amount of TFPI present.
TFPI antigen levels can be measured using an ELISA kit manufactured by American Diagnostica. The procedure is a sandwich-type assay using two anti-TFPI antibodies. Diluted plasma is incubated in a test well precoated with the initial capture antibody. A second indicator antibody is then added which binds to the captured TFPI. This second antibody has horseradish peroxidase enzyme attached. After washing, to remove unbound indicator antibody, an appropriate substrate is added and the amount of color produced is proportional to the amount of TFPI present.
It may well be that tests for these anticoagulants will never be
sensitive and accurate enough for routine use. Even if they are, the
fact still remains that trying to identify potential clotters would
require an extensive, ongoing screening program. This, as we all
know, is not financially feasible. It will probably be much more
effective to look for specific evidence of increased coagualtion
activity in patients with nonspecific signs of thrombosis.
This can be done by measuring;
or by measuring breakdown products or activation products of coagulation;
Prothrombin is a single amino acid chain. When it is converted to thrombin by activated X, a section of the chain, called the activation peptide, is cleaved from prothrombin. The remaining two fragments, one 36 amino acids long is linked by disulfide bonds to the remaining 259 amino acids to form thrombin. The activation peptide, which is now free in the plasma, is called Prothrombin fragment 1.2. A test is currently available for fragment 1.2 and if increased suggests invivo activation of the coagulation mechanism.
When factor X is activated by the factor VII/tissue factor complex, a piece of factor X is released called the factor X activation peptide. Tests are currently being devised to measure the presence of this fragment.
Factor VII can be activated by a number of different serine proteases, however, it is preferentially, and most effectively, activated by binding to tissue factor. Once activated factor VII has a relatively long half life, approximately 2 hours, compared to a half life of seconds to minutes for the other activated coagulation factors. This means that activated VII can be detected in the plasma of individuals experiencing a thrombotic event. Both immunologic and functional assays for Factor VII are available. The problem is trying to differentiate between the zymogem VII and activated VII.
In conclusion, what does this new understanding of the coagulation mechanism mean for the clinical laboratory? Tests of normal coagulation ability will continue to be performed. However, the recognition that relatively fewer patients die of hemorrhage, when compared to the number that experience thrombotic events, will result in smaller numbers of these tests. There will be an increase in tests (some described above) performed to identify inherited or acquired deficiencies in the anticoagulant system.