LeveL of overaLL hemostasis potentiaL in donor and patient pLasma in pathoLogy

Coagulation potential (CP), overall hemostasis potential (OHP) and fibrinolysis potential (FP) in plasma of donors and patients with myocardial infarction (MI), stroke (St) and hip joint diseases (hJD) have been investigated using M. Blomback’s global hemostasis assay. Plasma samples of the patients were analyzed with APTT reagent in the presence or absence of t-PA. It was found that the ratio of values of СP, OHP and FP in plasma of patients to those of donors plasma were 78, 60 and 123% at MI; 157, 155 and 162% at St; 128, 131 and 124% at HJD. CP to FP ratio that indicated balance between coagulation and fibrinolytic systems activities were 4.13, 2.5, 4.0 and 4.26 in plasma of donors and MI, St and HJD patients, respectively. These results are evidence of increased fibrinolytic activity in plasma of MI patients. Lag-periods of plasma clotting of MI, St and HJD patients were prolonged by 2.3, 7.2 and 1.5-fold, respectively. Pearson’s correlation analysis between parameters, obtained in vitro studies using global hemostasis assay, and concentrations of the molecular markers (soluble fibrin and D-dimer), which formed in vivo in plasma of MI, St and HJD patients, did not reveal any relationship between them.


Coagulation potential (CP), overall hemostasis potential (OHP) and fibrinolysis potential (FP) in plasma of donors and patients with myocardial infarction (MI), stroke (St) and hip joint diseases (hJD) have
been investigated using M. Blomback's global hemostasis assay.Plasma samples of the patients were analyzed with APTT reagent in the presence or absence of t-PA.It was found that the ratio of values of СP, OHP and FP in plasma of patients to those of donors plasma were 78, 60 and 123% at MI; 157, 155 and 162% at St; 128, 131 and 124% at HJD. CP to FP ratio that indicated balance between coagulation and fibrinolytic systems activities were 4.13, 2.5, 4.0 and 4.26 in plasma of donors and MI, St and HJD patients, respectively.These results are evidence of increased fibrinolytic activity in plasma of MI patients.Lag-periods of plasma clotting of MI, St and HJD patients were prolonged by 2.3, 7.2 and 1.5-fold, respectively.Pearson's correlation analysis between parameters, obtained in vitro studies using global hemostasis assay, and concentrations of the molecular markers (soluble fibrin and D-dimer), which formed in vivo in plasma of MI, St and HJD patients, did not reveal any relationship between them.

K e y w o r d s: hemostasis potential of blood plasma, soluble fibrin, D-dimer.
A global hemostasis assay developed by M. Blomback [1-8] is one of the methods of general characteristic of the plasma hemostatic system of donors and patients.It enables characterization of the functional activity of the coagulation and fibrinolytic systems, the relationship with each other as well as with concentrations of other hemostasis markers in patients' blood plasma.In particular, the method has been used to study the effect of different concentrations of FVIIa on global hemostasis and on TAFI (thrombin activated fibrinolysis inhibitor) dependent fibrinolysis in the blood plasma of patients with deficiency of FII, FV, FVII, FVIII, FIX, FXI and FXII [1].The authors believe that the method can be used for evaluation of the hemostatic effect of FVIIa, as well as for treatment with this factor of patients with hemophilia A and high level of anti-FVIIIa antibodies [2].It has been found that in patients with FXIIa deficiency the overall hemostasis potential and coagulation potential were increased, though fibrinolytic potential was significantly reduced, indicating higher risk of thrombosis [3].Another study reported that in patients with deep vein thrombosis associated with resistance to activated protein C, an increased procoagulant ac-tivity and an increased level of overall hemostasis potential were observed [4].The study on the effect of direct thrombin inhibitors such as dabigatran and argatroban on plasma clotting using the global hemostasis assay showed that these inhibitors reduce the coagulation potential and enhance the fibrinoly tic potential of blood plasma [5].The assay was also used to examine the blood plasma of patients with various diseases accompanied by hemostatic system disorders, namely arterial and venous thromboembolism, coronary artery disea se, ischemic heart disea se, autoimmune diseases, etc. [914].
In this paper we present the results of determination of the CP, OHP and FT values, as well as the relationship between these indicators and their relationship with the concentrations of such hemostatic molecular markers as soluble fibrin and Ddimer in the plasma of donors and patients with hip joints disea ses, myocardial infarction and stroke immedia tely after admission to the hospital.

APTT (activated partial thromboplastin time)
reagent (RENAM, Russia), recombinant tissue plasminogen activator (tPA) (Boehringer Ingelheim, doi: http://dx.doi.org/10.15407/ubj88.02.056Germany) were used in the study.Blood of donors and patients were sampled in 3.8% sodium citrate (1 part of sodium citrate and 9 parts of blood, pH 7.4).Patients' blood sampling was performed before treatment.Plasma was separated from blood cells within 1 h after blood sampling by centrifugation at 3000 g for 20 min.Plasma aliquots were stored at -20 °C.
Hemostasis potential in plasma was determined using spectrophotometry recording the absorbance by the fibrin clot at 405 nm on microreader Multiscan (Finland).The clots were formed in wells of microplate, to which the following reagents were added sequentially: 0.05 M HEPES buffer (pH 7.4), containing 0.15 M NaCl, 70 µl blood plasma, tPA to a final concentration of 75 IU/ml and APTT reagent.The plasma clotting was initiated by adding of 25 mM CaCl 2 .The final reaction volume was 300 µl.OHP is characterized by the area under the curve of clot turbidity from the initiation point of plasma coagulation until the complete clot degradation in the presence of t-PA.CP is the area under the curve of clot formation from plasma coagulation initiation until a moment of the complete clot degradation in the absence of tPA.FP is the difference between the values of CP and OHP.All values are expressed in units of absorbance multiplied by the time in seconds (a.u.×s) [14].
The concentrations of fibrinogen, soluble fibrin and D dimer in blood plasma were determined by an enzyme immunoassay using the test systems developed at the Palladin Institute of Biochemistry of NAS of Ukraine [15].Statistical analysis was performed using standard Excel software.Average values and their standard deviation were calculated, and correlation pair analysis was performed.

results and discussion
To characterize the hemostatic system in blood plasma of donors and patients, we determined parameters of blood coagulation curve that describe the coagulation process on the whole and its main stages [16].The plasma ability to form and degrade the clot in the presence of APTT reagent and t-PA was evaluated by assessment of the hemostasis potential, which comprises three components, namely the CP that characterizes the work of the intrinsic pathway of the coagulation cascade; FP that charac terizes the ability of the fibrinolytic component of the system to degrade the clot; OHP that characteri zes the ability of the hemostasis system to maintain the balance bet ween the formation and degradation (lysis) of blood plasma clot.The separate stages of coagulation process were determined by such parameters as lag period, which indicates the rate of coagulation cascade activation, formation of thrombin and protofibrils; the maximum rate of increase in turbidity, which indicates the rate of protofibrils lateral association and fibril formation; the clot turbidity that reflects the clot fiber thickness [17].Moreover, the ratio between the values of CP, OHP and FP as well as the concentrations of hemostasis molecular markers (fibrinogen, soluble fibrin, Ddimer) were presented in this study.The average values of studied parameters for donors' blood plasma are shown in Table 1.
Typical curves for the blood plasma coagulation of donors are shown in Fig. 1.
It is seen that the curves on the interval of plasma clotting coincide for both systems.At that clot turbidity quickly reached a maximum and in the absence of tPA did not further change, that is, clot formation was completed.Since the rate of protofib ril lateral association and fibril formation did not alter in the presence of tPA, the formation of fibrinolytic enzyme plasmin, which would degrade the fibrin polymer, did not occur during the interval of reaching the plateau of the curve (Fig. 1) [18].It is known that the protofibrils binding with fibril competes with the binding of plasminogen (Pg) and tPA with the fibril surface, and hence Pg and tPA are observed only on the fibril surface of the formed clot [19].After reaching plateau, which indicates the completion of the fibril clot formation, activation of the fibrinolytic system and degradation of clot structure begin.However, the turbidity decreased slowly owing to cleavage of mainly fibrin αCfragments at this stage [20,21].After the Cterminal lysine appeared on fibrin molecules, the Pg activation enhanced significantly and the clot degradation accele rated and quickly completed (Fig. 1) [22,23].

Fig. 1. Typical curves for the blood plasma coagulation of donors induced by APTT reagent in the presen ce and absence of t-PA
The curves obtained for plasma clot formation and degradation in the presence of t-PA upon all studied diseases had similar shape with certain differences.
First of all, they differ in the duration of the lag period, which is extended upon all studied diseases.Thus, at stroke the rate of coagulation pathway activation as well as protofibril formation slowed down 7fold (Fig. 3, Table 1).This might be associated with the release of heparin in the bloodstream and the formation of its complex with antithrombin III.It was also observed that the maximum clot turbidi ty increased 1.5-fold upon stroke.The maximum clot turbidity at myocardial infarction was on the avera ge 25% smaller in the presence of tPA than that in the absence of tPA as well as in donors' plasma (Fig. 2, Table 1).The curves for blood plasma coagulation in patients with HJD and donors did not differ from each other in shape (Fig. 4 and Fig. 1).The clot turbidity in the blood plasma of these patients, compared to the norm, was smaller (Table 1).Clot half-lysis time increased upon all studied diseases that indicated an increase in the plasma fibrinolytic inhibitors concentration (Table 1).
Curves for alterations in clot turbidity during the blood plasma clotting were used to determine general parameters of the process, namely hemostasis potential and its components such as CP, OHP and FP which indicate formation rate, life time and fibrin clot lysis rate in blood plasma in vitro.
Blood plasma samples (25 samples) of patients with myocardial infarction who had just been admitted to the hospital were examined.It was found that CP and OHP reduced by 78 and 60%, respectively, compared to donors, and FP was 123% of the norm (Table 1).This indicates that in these patients the balance between coagulation and fibrinolysis shifted towards the latter.Lag period of plasma coagulation increased, indicating the inhibition of thrombin formation rate in the intrinsic pathway in vitro.Level of the hemostasis molecular markers in vivo was found to be: Fg remained within the normal range, the concentrations of soluble fibrin and Ddimer increased by 57 and 72%, respectively.
The potentials values of plasma hemostatic system in patients with a stroke.Blood plasma samples of patients with a stroke (10 samples) were studied.In these patients, CP and OHP were greater than that in donors by 57 and 55%, respectively (Table 1).FP increased by 63%.Moreover, a significant almost 7fold increase in the lag period, compared to control plasma, was observed.Upon this disease we observed a paradoxical situation in which the inhibition of activation of the coagulation intrinsic pathway, accompanied by a decrease in thrombin concentra-tion, led to an increase in the rate of fibrin clot formation as well as to significant increase in the clot turbidity and the time of its existence, despite the increase in the plasma FP (Table 1).This situation might be related to an increase in inhibitor concentrations in the intrinsic pathway of coagulation that inhibits the thrombin formation.At a lower concentration of thrombin, longer fibrin protofibrils, which parame ters, which had been obtained in two experimental systems, were studied.Blood plasma was one of the systems, in which parameters of the fibrin clot formation and degradation in vitro under the influen ce of APTT reagent in the presence or absence of tPA were studied.In another system, hemostasis molecular markers that were formed in blood plasma in vivo and characterized the hemostasis state of donors or patients were studied using immunochemical testsystems [15].Point of interest was to study the relationship between the parameters within each system as well as between these two systems.The revealing of such connection would allow charac terization of the state of homeostasis in vivo based on the parameters of the clot formation process in the blood plasma in vitro.The values of Pearson's linear correlation coefficients for a pair of parameters, the relationships between which are not random (the c.c./c.cor.ratio ≥ 3.0) are represented in Table 2.

T a b l e 1. Hemostasis potential, coagulation parameters and concentrations of fibrinogen, soluble fibrinogen and D-dimer in blood plasma activated by APTT reagent of donors (n = 10) and patients. A -in the absence of t-PA; B -in the presence of t-PA; C -hemostasis potential parameters at norm and diseases; Dmolecular markers concentrations
A correlation analysis of the plasma parameter values in patients with MI in vitro at plasma activation by APTT reagent revealed the closest relationship between the following parameters: CPOHP, CPFP, OHPH, CPH, where the correlation coefficients were 0.97, 0.82, 0.92 and 0.84, respectively.The correlations between the parameters of CP OHP and OHPH are illustrated in Fig. 5. Weaker relationship was observed between FPH and CPα, OHPα, the correlation coefficients were 0.6 and 0.74, 0.70, respectively.The first value indicates a weaker relation between FP and the clot turbidity.The correlation coefficient for the pair CP and OHP demon strates that these potentials are more related to the clot turbidity than to the rate of the protofibril lateral association at the clot formation.Among the hemostasis blood plasma molecular markers determined by immunochemical method, the correlation between Fg and soluble fibrin was revealed.The correlation between concentrations of fibrinogen and Ddimer was not observed.The obtained value for correlation coe fficient between the concentrations of soluble fibrin and Ddimer did not reliably indicate a relationship between these parameters.The relation between system parameters in vitro and molecular markers that are formed in vivo were also not revealed.
Analysis of correlation between plasma parame ters of patients with St and HJD revealed, similar to MI patients, the relationship between values of the parameters in the plasma samples activated by APTT reagent in vitro (Table 2 and Fig. 6).are laterally associated with a higher rate, are formed [24].An increase in the maximum clot turbidity is obviously related to the inclusion of plasma proteins such as fibronectin, von Willebrand factor, albumin to the clot [25,26].On the other hand, analysis of the ratio between CP and FP revealed that the balan ce between coagulation and fibrinolysis in patients approached normal physiological values, 4.58 and 4. 13, respectively.The level of soluble fibrin was higher by 42% than the norm, whereas the level of D dimer was higher by 274% compared to the norm that indicates the high activity of blood plasma fibrinolysis in vivo.
The plasma hemostasis potentials in patients with hip joint disease.Blood plasma samples (8 samples) of patients with HJD were investigated.It was found that CP and OHP values were by 28 and 31% higher than normal values.FP increased by 24%.However, the CP/FP ratio that characterizes the balan ce between coagulation and fibrinolytic systems shifted significantly toward the coagulation system by 68% compared to the norm.Fibrinogen level was in the normal range.Though, the level of soluble fibrin was slightly (by 27%) higher than normal value, and Ddimer was higher by 85% indicating that the fibrinolytic system activity is sufficient for maintaining a balance between these systems in vivo.
The correlation between the hemostasis parameters and the concentrations of the hemostasis markers.The two groups of the hemostatic system Concentrations of molecular markers formed in blood plasma in vivo altered independently.Correlations between the plasma coagulation parameters in the presence of APTT reagent and concentrations of hemostasis molecular markers in vivo were also not found.

Hemostasis parameter Correlation coefficient
Thus, the study of hemostasis potential in plasma samples of donors and patients with MI, St and HJD showed that the blood plasma OHP value in MI patients decreased 1.7 times, while in patients with St and HJD increased 2.1 and 1.7 times, respectively (Table 1).A similar tendency was also observed for the blood plasma CP.The blood plasma FP increased upon all diseases.
However, CP/FP ratio, which reflects the balan ce between coagulation and fibrinolytic systems in the patients' blood plasma, indicates that imbalance occurs only in MI patients.
The analysis of the linear correlation between the parameters showed that the relationship exists only in artificial plasma system in vitro activated by APTT reagent.Correlation analysis of the rela-

Fig. 2 .Fig 3 .
Fig. 2. Typical curves for the blood plasma coagulation induced by APTT reagent in the presence and the absence of t-PA of patients with a myocardial infarctionTime, s

Fig. 4 .
Fig. 4. Typical curves for the blood plasma coagulation induced by APTT reagent in the presence and absence of t-PA of patients with hip joint disease