Th.--Méd.--Montpellier, 1912-1913. / Montpellier, 1912-1913, n ° 73.

Th.--Méd.--Nancy 1, 1984. N°: 307.

The main aims of this work were to develop and test methods for the analysis of the shape and calcium binding properties of fibrinogen and its degradation product fragment D. The techniques of photo-sensitized labelling and cross-linking were used to obtain information about the shape of these proteins and both revealed interesting information. Photosensitized labelling proved particularly useful and results obtained using this technique suggested that fragment D (Ca++) in solution and the fragment D domains of fibrinogen are conformationally very similar. These studies also suggested that the A chain of fibrinogen is highly exposed at the surface of the molecule and that it shields other portions, particularly the D-domains of the molecule. The use of chemical cross-linking reagents revealed a gross-conformational difference between fragment D (Ca++) and fragment D (EDTA) and it was further shown that this difference is a result of cleavage of the chain of fragment D (Ca++) rather than calcium removal. This suggests that the calcium ion bound by fragment D (Ca++) exercises a protective influence over a plasmin susceptible bond and that cleavage of this bond allows a gross conformational change. This is consistent with the results from photo-sensitized labelling which showed the C-terminal portion of the chain of fragment D to the surface exposed. It was also shown that the technique of photo-sensitized labelling could be modified to induce cross-linking yielding a plasmin digestable product. These studies confirmed results from labelling studies which suggested that the chain C-terminii protect the N-terminal portion of fibrinogen. Calcium binding studies revealed two classes of high affinity calcium binding site in fibrinogen. Two sites of high (Kd > 10-5m) were shown to exist in the D-domains of the molecule, a third site of even higher affinity (Kd > 10-7m) was also found although the position of this site was not identified. These results of the study may be used to reconcile some of the more extreme views of fibrinogen shape as they suggest that fibrinogen is a protected trinodular structure.

There is much controversy regarding the conformation of fibrinogen. Several models have been proposed ranging from a trinodular arrangement to a globular conformation. It has also been suggested that fibrinogen has a flexible structure where the shape of the molecule is influenced by its environment, one major factor being calcium concentration, In addition, although the importance of tightly bound calcium ions (kd 1M) to the fibrinogen molecule is well established, the role of the larger number of low affinity sites (kd 1mM) is still a matter of some debate. In this study, the two techniques of photosensitized radioactive surface labelling and photosensitized cross-linking were selected for development and assessment. This was done with a view to examining the conformation of fibrinogen in its native state and under different solvent conditions, with particular reference to the influence of calcium. The two techniques have been shown to have definite applications in their use as probes into the conformation of fibrinogen. Results derived using these methods support the view put forward by various authors that fibrinogen is a dynamic molecule having a flexible conformation and that the conformation adopted is dependent on solvent composition. Calcium concentration in the millimolar range is particularly significant and consequently so is the saturation of the low affinity binding sites which may well have a regulatory function. Experimentally two extreme conformations have been demonstrated, - a closed, compact structure at low calcium concentrations compared to a more open one at higher calcium concentrations. More importantly, the techniques used also show the subtle changes which occur within the molecule as the calcium concentration is raised, changes which may be more significant physiologically. The most important of these are the effect of calcium on the C-terminal parts of the Aa-chains and the D domains of the molecule.

A purification method to obtain a fibrinogen preparation with a high proportion of intact molecules and free from contaminating plasminogen and Factor XIII was developed. Such fibrinogen preparations were used in cross-linking and plasmin-digestion studies to investigate the possible involvement of the carboxyl terminal regions of the A-chain in a high affinity calcium-binding site. The results of these investigations gave no evidence to support such a role for the carboxyl terminals of the A-chains. Highly intact fibrinogen preparations were also used in experiments to re-assess the number of high affinity calcium-binding sites in the molecule. The technique of flow dialysis was employed. Results from these experiments were in agreement with previously published data that there are three high affinity calcium-binding sites in fibrinogen. Indirect evidence was obtained which suggests that the chelator EGTA binds to the fibrinogen molecule.

(1) Three molecular weight forms of fragment D were isolated from a plasmic digest of human fibrinogen. Further heterogeneities within the preparation were revealed by NH2-terminal amino acid analysis and by digestion studies performed in the presence of 2 N-urea; the existence of conformationally different forms of the fragment D molecule is proposed. (2) Plasmic digestion of fibrinogen in the presence of 2 md-Ca2+ produced a homogeneous high molecular weight preparation of fragment D (designated DCa 2+) In the absence of Ca2+ or in the presence of EDTA, two lower molecular weight forms of fragment D, each containing a more degraded constituent γ chain, were identified. In the presence of 2 N-urea only slight plasmic degradation of fragment DCa2+ occurred. (3) Increased SDS-gel electrophoretic mobilities were demonstrated for the fragments D and Y prepared from fibrinogen in the presence of Ca2+. An anomalous electrophoretic mobility pattern was also described for the constituent γ chain of fragment DCa2+ and of fibrinogen exposed to Ca2+. It is suggested that Ca2+ bound to the constituent γ chain of fibrinogen and fragment D forms an intra chain Ca2+ -bridge towards the COOH-terminus thereby maintaining a 'hook-like' conformation. This form of fragment D, it is proposed, exhibits a decreased susceptibility to plasmic attack and an anomalously low electrophoretic mobility. (4) A method was developed employing ion-exchange chromatography and gel filtration for the preparation and isolation of fragment D both in the presence and absence of free Ca2+. (5) Studies employing purified samples of each type of fragment D confirmed that the two molecules differed in the extent of degradation of the constituent γ chain at the COOH-terminus. Furthermore an influence of Ca2+ on the charge heterogeneity of fragment D preparations was concluded from isoelectric focussing studies. (6) The proposal of a compact and thereby stable structure for the fragment DCa2+ molecule was strengthened by the results obtained from studies employing chemical crosslinking reagents and the technique of ultracentrifugation. (7) The possibility that serine, glycine and glutamate amino acid residues are located in the region of the γ chain associated with the binding of Ca2+ was suggested from amino acid analysis of fragment D.

Haemostasis is the result of a fine balance of interactions between the endothelium, plasma proteins and blood cells under a wide range of flow rates. To mimic these conditions in vitro, a parallel plate flow chamber with human umbilical vein endothelial cells (HUVEC) or extracellular matrix (ECM) has been developed. A method to investigate thrombin generation (TG) in platelet rich defibrinated plasma was validated and used to investigate inhibition by two distinct classes of antithrombotic agents: anti-platelet antibodies and anticoagulants, including unfractionated heparin (UFH), low molecular weight heparin (LMWH) and hirudin. Increasing flow rates increased TG, which was higher in the presence of ECM than in the presence of HUVEC. All anti thrombotic agents investigated were less effective in the presence of ECM. The monoclonal anti-platelet glycoprotein (GP) lIb/IlIa antibody, RFGP56, partially inhibited TG under static or arterial flow conditions and was less effective under venous flow conditions. The monoclonal anti-platelet GP Iba antibody, RFGP37, did not inhibit TG under flow or static conditions. A combination of the two antibodies showed no further activity than RFGP56 alone. UFH, which has equal anti-factor Xa and anti-thrombin activity, was able to inhibit TG under static and flow conditions. On an anti-Xa unit basis, comparatively more LMWH (with a 10: 1 ratio of anti-factor Xa to anti-thrombin activity) was required to inhibit TG under static and venous conditions, but under arterial conditions LMWH was as effective as UFH. Hirudin, a thrombin specific inhibitor, was totally effective under static conditions, but was only able to inhibit up to 40 % ofTG under flow. This study shows that some anti-platelet agents can inhibit coagulation and this may contribute to their antithrombotic efficacy under certain flow conditions. Although both the anti-factor Xa and anti-thrombin activities of anticoagulants are effective, anti-factor Xa activity may play a more important inhibitory role under flow conditions.

Introduction: Haemostasis is the arrest of bleeding. In recent years, in-vitro studies have suggested that secondary haemostasis (blood coagulation) is subject to activation by reactive oxygen species (ROS). It is known that patients who suffer from vascular disease are typically hypoxaemic and in the case of peripheral occlusive artery disease (POAD), physical exercise is used to improve symptom free mobility in the affected limbs. Hypoxia and physical exercise are two potent independent and synergistic initiators of ROS. We identified a clear need for in-vivo analysis of this novel area of research. Aims: There were two main aims of this research. 1. To explore the in-vivo influences of inspiratory hypoxia and physical exercise on biomarkers of haemostasis; and in doing so and subsequently carry out a randomised double blind placebo control trial to explore the interaction between oxidative stress (ROS accumulation) and haemostasis. Hypothesis: It was hypothesised that hypoxia and exercise would be independently and synergistically associated with an increase in oxidative stress, resulting in coagulation activation. We hypothesised that intervention with free radical reaction-chain breaking antioxidant vitamins would attenuate oxidative stress and thus attenuate the activation of coagulation. Methods: study 1 - Healthy males were subjected to six hours of normobaric hypoxia (12% inspired oxygen) and then a physical exercise challenge to exhaustion (cycling ramp-test). Citrated plasma was collected pre hypoxic exposure, post six hours of exposure, then immediately post exercise and analysed for routine clinical markers of coagulation (aPTT, PT, TT and fibrinogen) and analysed with and without correction for plasma volume shift. Data were analysed using a one-factor repeated measures ANOVA incorporating one within (condition: time point) subjects factor. Following a significant main effect and interaction, paired samples t-tests were employed to make post hoc comparisons at each level of the within-subjects factor. Study! - Healthy males were subjected to a double blind, randomised, placebo controlled intervention with vitamin C (a water soluble) and vitamin E (a lipid soluble), two ROS-scavenging, chain breaking antioxidants. The intervention lasted eight weeks to insure membrane enrichment with antioxidants. The methods of study one were repeated but with a pre-intervention time point added and the addition of two extra markers of thrombin generation (PF1+2 and T-AT). Data were analysed using a two-factor mixed ANOVA incorporating one between (group: antioxidant intervention vs. placebo control) and one within (condition: time point) subjects factor. Following a significant main effect and interaction, a paired samples t-test was used to make post hoc comparisons at each level of the within-subjects factor, with the alpha level Bonferroni corrected for multiple comparisons Between-group comparisons were assessed using independent samples t-tcsts applied to each level of the between-subjects factor. Results: Study 1 - Hypoxia was not associated with activation of coagulation. Physical exercise increased the activity of contact factor coagulation pathway activation. Study 2 - The intervention increased thrombin generation in the antioxidant group. This was met with an antagonistic antithrombin activation. Hypoxia did not impact the placebo group, but normalised the thrombin generation of the antioxidant group. Physical exercise increased contact factor pathway (CFP) as per study 1, but thrombin generation was unaltered. Hypoxia suppressed fibrinolysis post exercise, which is known to be activated in normoxic exercise. Discussion: hi study one, hypoxia alone did not activate coagulation. We hypothesised that this could be tentative evidence of a ROS concentration threshold for activation since once exercise was superimposed the accumulation of ROS activated the CFP. Correction for changes in plasma volume nullified the increased activity of the CFP. Corrections for shifts in plasma volume are routinely ignored in the literature and this was a novel finding. Study 2 was the first intervention of its kind. The increase in thrombin generation pre-post intervention with antioxidants suggests compelling evidence of in-vivo regulation of coagulation by ROS. But the direction of change was completely contrary to the original hypothesis. The confirmation that hypoxia does not activate coagulation is important, especially given the controversy surrounding long-haul flight deep vein thrombosis. Interestingly, exercise did not increase thrombin generation, despite the increase in the CFP. These findings suggest haemostasis is indeed subject to control by the body's redox state invivo via an as of yet, unknown mechanism.

M.Tech. / Coagulation or the formation of blood clots is the result of several complex interactions between humoral coagulation factors, platelets, and fibrin. Unnatural or excessive coagulation is inhibited by the fibrinolytic system. In normal homeostasis, there is a dynamic state in which thrombi are constantly being formed and removed from the circulatory system. Fibrin being the main component of a blood clot is formed by the activation of the clotting cascade. Its production is followed by the fibrinolytic system, resulting in plasmin generation and subsequent fibrin degradation (Soria et al., 1983). Plasmin is the enzyme responsible for fibrin degradation. It is derived from its inactive precursor, plasminogen, by the action of thrombin and plasminogen activators. Homoeopathic Arnica montana is prescribed for pathological conditions that have a sudden onset, are traumatic in nature, and result from the complications of the initial trauma (Vermeulen, 1997). It has been prescribed for various thrombotic disorders and it is known that it speeds up healing and revascularisation of the surrounding tissue (Savage and Roe, 1977). It is a short acting homoeopathic medication, and needs to be repeated often, but it is quick in its actions (Gordon Ross, 1977). This study aimed to establish if Arnica montana 30CH and 200CH potencies caused thrombolysis of a blood clot within an in-vitro sample. It was hypothesised that Arnica montana in 30CH and 200CH potencies would cause thrombolysis in- vitro. The research sample group consisted of fifteen male participants, between eighteen and thirty years of age. Only male participants were selected to prevent any gender variables that may influence the study. Four blood samples each consisting of five milliliters was taken from each participant. Current and efficient phlebotomy techniques were used and the samples were placed in non-treated plastic phlebotomy containers to allow speedy clot formation. One sample was treated with one drop of 30CH Arnica montana and the other with one drop of 200CH Arnica montana. The third sample for each subject was used as a control where one drop of 0.9% sterile saline was added. Thus, consistency concerning diluting effects was maintained. D-dimer levels were measured using the D-DI Test from Diagnostica Stago, which is a rapid latex agglutination slide test. A semi-quantitative testing mode was used to gather the relevant research information. The results of the study showed that in all the samples tested, no agglutination of D-dimers occurred. This indicated that if thrombolysis occurred in the samples, a D-dimer level well below 0.5 micrograms per milliliter of FEU occurred. It has been established that as separate entities homoeopathic Arnica montana 30CH and 200CH have little or no direct effect on a clotted in-vitro sample. This indicates that if the medicine has thrombolytic properties, it has to utilize or activate other endogenous factors in-vivo. As these factors require the presence of vascular endothelium, it makes it potentially difficult to conduct studies due to ethical reasons.

M.Tech. (Homoeopathy) / The purpose of this study was to determine the effect of the Homoeopathic remedy Arnica montana 12 CH on blood coagulation. For years Arnica montana has been used to treat injuries where there is bleeding and bruising. The arrest of bleeding is accomplished by a number of mechanisms,one of which is blood coagulation. The study was conducted using 21 volunteers with each person acting as his or her own control Two blood samples were collected from each patient over a three day period. After preparing fresh whole blood samples and platelet-poor plasma, full blood count and coagulation assays were run using the Cell-Dyn 1700 system and the Automated Coagulation Laboratory analyser respectively. The average from the two days were calculated and statistically analysed using the Two way ANDVA method of analysis. Results showed that the differences between the experimental group and the control group were too small to be statistically significant. This showed that Arnica montana 12 CH had no significant effect on blood coagulation. It is interesting to note that the alcohol group had almost the same results as the experimental group.