An enhanced clot growth rate before in vitro fertilization decreases the probability of pregnancy.

BACKGROUND: The shift towards hypercoagulation during in vitro fertilization (IVF) can lead to the impairment of embryo implantation and placental blood circulation, which is believed to be a factor in an unsuccessful IVF cycle. OBJECTIVES: To assess coagulation in women with infertility before the start of an IVF cycle and during treatment to reveal the association between coagulation imbalance and IVF outcome. PATIENTS/METHODS: We conducted a prospective cohort observational study including 125 participants who underwent fresh IVF cycles. Blood samples were collected at five time points: before IVF, one week after the start of controlled ovarian stimulation (COS), on the day of follicular puncture, on the day of embryo transfer (ET) and one week after ET. Coagulation tests (clotting times: activated partial thromboplastin time (APTT) and prothrombin; fibrinogen and D-dimer concentrations; thrombodynamics) were performed. RESULTS: Women with an elevated clot growth velocity (>32.3 µm/min, detected by thrombodynamics) before IVF demonstrated a higher risk of negative IVF outcomes (adjusted RR = 1.38; 95% CI 1.28-1.49; P<0.001). During the procedure, we observed increases in prothrombin, fibrinogen and D-dimer concentrations, a slight shortening of APTT and a hypercoagulation shift in the thrombodynamics parameters. The hemostasis assay values during COS and after ET had no associations with IVF outcomes. CONCLUSIONS: Hypercoagulation in the thrombodynamics before the start of IVF treatment was associated with negative IVF outcomes. After the start of COS, all tests demonstrated a hypercoagulation trend, but the hypercoagulation did not influence IVF outcome. This research is potentially beneficial for the application of thrombodynamics assay for monitoring hemostasis in infertile women prior to an IVF procedure with the goal of selecting a group requiring hemostasis correction to increase the chances of pregnancy.

Alterations in the parameters of classic, global, and innovative assays of hemostasis caused by sample transportation via pneumatic tube system.

BACKGROUND: Pneumatic tube system (PTS) is an integral part of large medical facilities providing rapid interconnection between units within the hospital and often used to transport blood samples. The aim of our study was to compare a wide variety of hemostasis assays to identify assays sensitive to this transport method and diagnostic relevance of the alterations. METHODS: Routine coagulation and platelet tests (APTT, PT, TT, fibrinogen, light transmission aggregometry (LTA) with ADP, collagen, ristomycin and epinephrine), whole blood flow cytometry platelet function test (levels of CD42b, CD61, CD62P, PAC1, annexin V binding and mepacrine release) and global coagulation tests (thromboelastography (TEG), thrombin generation (TGT), thrombodynamics (TD), thrombodynamics-4D (TD-4D)) were determined in PTS- and manually transported samples of 10 healthy volunteers. RESULTS: There were no significant differences between the values of APTT, PT, TT or fibrinogen between the samples transported by PTS or manually. The results for LTA demonstrated increase in the collagen-induced aggregation (84 ±â€¯7% versus 73 ±â€¯5%), while the response to epinephrine was decreased (58 ±â€¯20% versus 72 ±â€¯7.4%). Flow cytometry-based platelet function test showed a pre-activation of platelets by PTS-transportation while all integral assays of coagulation tested in the present study (TEG, TGT, TD, TD-4D) demonstrated a hypercoagulation shift. CONCLUSIONS: Transportation by PTS caused significant shifts in parameters of functional and integral assays that exceeded parameter variation values and sometimes even were comparable to normal ranges. The results obtained in this study indicate that using of PTS for such assays may cause sufficient alterations of results and can lead to patient's mistreatment.

Eculizumab effect on the hemostatic state in patients with paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by a hypercoagulable state associated with acute hemolysis. Eculizumab is used to reduce the intensity of intravascular hemolysis in PNH patients. The hemostatic status of three patients with PNH was assessed during eculizumab treatment by D-dimer assay and the global assays: thromboelastography (TEG), thrombin generation test (TGТ), and thrombodynamics (TD). In the state of hemolytic crisis before the therapy D-dimer concentration was increased in two patients accompanied by hypercoagulation changes in TEG parameter angle (α). TD parameter the clot growth velocity (V) revealed hypercoagulability while TGT parameter ETP was within the normal range in all patients. The lactate dehydrogenase (LDH) activity decreased during the 8months of eculizumab therapy. The physical health was improved, the frequency of hemolytic crisis decreased. Patients periodically exhibited hypercoagulable state: the mean values α=38±11° (with normal range 20-40°), ETP=1311±442nM·min (with normal range 800-1560nM·min), V=31±4µm/min (with normal range 20-29µm/min). During the eculizumab therapy two patients had the repeated clinical manifestation of acute hemolytic crisis, the parameters of the global tests were increased compared to the previous measurement. The global hemostasis tests TEG, TGT and TD revealed hypercoagulability in patients with PNH during eculizumab therapy.

[Influence of temperature on spatial fibrin clot formation process in thrombodynamics].

In this study we have investigated the process of spatial fibrin clot formation in non-steered platelet-free plasma at the temperatures from 20°C to 43°C using thrombodynamics - the novel in vitro hemostasis assay, which imitates the process of hemostatic clot growth in vivo. During data processing the following parameters were calculated: initial (V i ) and stationary (V st ) rates of clot growth which characterize initiation and propagation phases of clotting process, and clot size on the 30 th minute. The temperature dependence of extrinsic and intrinsic tenase activities, which determine values of the initial and stationary clot growth rates, respectively, have been also measured. It was established that the temperature lowering from 37°C to 24°C extends mainly on the initiation phase of clot growth, while the stationary rate of clot growth changes insignificantly. Meanwhile none of the thrombodynamics parameters shows the dramatic change of plasma coagulation system condition at the temperature of 24°C (acute hypothermia). Using the thrombodynamics assay an assumption, that the temperature lowering does not change the state of plasma hemostasis system significantly has been confirmed.

Thrombin activity propagates in space during blood coagulation as an excitation wave.

Injury-induced bleeding is stopped by a hemostatic plug formation that is controlled by a complex nonlinear and spatially heterogeneous biochemical network of proteolytic enzymes called blood coagulation. We studied spatial dynamics of thrombin, the central enzyme of this network, by developing a fluorogenic substrate-based method for time- and space-resolved imaging of thrombin enzymatic activity. Clotting stimulation by immobilized tissue factor induced localized thrombin activity impulse that propagated in space and possessed all characteristic traits of a traveling excitation wave: constant spatial velocity, constant amplitude, and insensitivity to the initial stimulation once it exceeded activation threshold. The parameters of this traveling wave were controlled by the availability of phospholipids or platelets, and the wave did not form in plasmas from hemophilia A or C patients who lack factors VIII and XI, which are mediators of the two principal positive feedbacks of coagulation. Stimulation of the negative feedback of the protein C pathway with thrombomodulin produced nonstationary patterns of wave formation followed by deceleration and annihilation. This indicates that blood can function as an excitable medium that conducts traveling waves of coagulation.

Positive feedback loops for factor V and factor VII activation supply sensitivity to local surface tissue factor density during blood coagulation.

Blood coagulation is triggered not only by surface tissue factor (TF) density but also by surface TF distribution. We investigated recognition of surface TF distribution patterns during blood coagulation and identified the underlying molecular mechanisms. For these investigations, we employed 1), an in vitro reaction-diffusion experimental model of coagulation; and 2), numerical simulations using a mathematical model of coagulation in a three-dimensional space. When TF was uniformly immobilized over the activating surface, the clotting initiation time in normal plasma increased from 4 min to >120 min, with a decrease in TF density from 100 to 0.7 pmol/m(2). In contrast, surface-immobilized fibroblasts initiated clotting within 3-7 min, independently of fibroblast quantity and despite a change in average surface TF density from 0.5 to 130 pmol/m(2). Experiments using factor V-, VII-, and VIII-deficient plasma and computer simulations demonstrated that different responses to these two TF distributions are caused by two positive feedback loops in the blood coagulation network: activation of the TF-VII complex by factor Xa, and activation of factor V by thrombin. This finding suggests a new role for these reactions: to supply sensitivity to local TF density during blood coagulation.

Improvement of spatial fibrin formation by the anti-TFPI aptamer BAX499: changing clot size by targeting extrinsic pathway initiation.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is a major regulator of clotting initiation and a promising target for pro- and anticoagulation therapy. The aptamer BAX499 (formerly ARC19499) is a high-affinity specific TFPI antagonist designed to improve hemostasis. However, it is not clear how stimulation of coagulation onset by inactivating TFPI will affect spatial and temporal clot propagation. OBJECTIVE: To examine the BAX499 effect on clotting in a spatial, reaction-diffusion experimental system in comparison with that of recombinant activated factor VII (rVIIa). METHODS: Clotting in plasma activated by immobilized tissue factor (TF) was monitored by videomicroscopy. RESULTS: BAX499 dose-dependently improved coagulation in normal and hemophilia A plasma activated with TF at 2 pmole m(-2) by shortening lag time and increasing clot size by up to ~2-fold. The effect was TFPI specific as confirmed by experiments in TFPI-depleted plasma with or without TFPI supplementation. Clotting improvement was half-maximal at 0.7 nm of BAX499 and reached a plateau at 10 nm, remaining there at concentrations up to 1000 nm. The BAX499 effect decreased with TF surface density increase. RVIIa improved clotting in hemophilia A plasma activated with TF at 2 or 20 pmole m(-2) , both by shortening lag time and increasing spatial velocity of clot propagation; its effects were strongly concentration dependent. CONCLUSIONS: BAX499 significantly improves spatial coagulation by inhibiting TFPI in a spatially localized manner that is different to that observed with rVIIa.

Thromboplastin immobilized on polystyrene surface exhibits kinetic characteristics close to those for the native protein and activates in vitro blood coagulation similarly to thromboplastin on fibroblasts.

A method for transmembrane protein thromboplastin (tissue factor) immobilization on polystyrene surface is described. Tissue factor is the main activating factor launching the blood coagulation process. It is a cofactor of factor VIIa, the first protease in the cascade of coagulation reactions. The proposed method preserves kinetic characteristics specific for native tissue factor on the fibroblast surface. The kinetics of binding to factor VIIa and enzymic activity of the formed complex follow Michaelis-Menten kinetics, which is also characteristic of native complex. A small difference is that dissociation constant for tissue factor immobilized on polystyrene surface exceeds 2.7-fold that for native factor. The proposed technique of immobilization provides for protein density on the activating surface corresponding to the tissue factor density on the fibroblast surface. The immobilized tissue factor can be used to activate blood coagulation in methods simulating spatial dynamics of in vitro clot growth. Investigation in this direction will make it possible to register both hypo- and hypercoagulation states of the system. This approach is advantageous over traditional methods of estimation of the coagulation system conditions, which mainly register only hypocoagulation. Investigation of the storage time has shown that activators containing immobilized tissue factor can be stored and used during for at least 100 days in the method studying spatial dynamics of fibrin clot formation.