Blood Clot Contraction Dynamics Studied with an Automated Analyzer System

Blood clot contraction plays an important role in hemostasis because it seals a vessel injury, approximates wound edges and restores blood flow past obstructive thrombi. The process is governed by contraction of activated platelets and requires the interaction of platelets with fibrin, however, many aspects of this process are not fully understood. Despite the biological and medical importance, clot contraction has been systematically studied neither in research nor in clinical laboratories. One reason for that is the lack of methodology to follow and quantify clot contraction dynamics. We have developed a novel method to assess blood clot contraction in vitro and successfully applied this technique to investigate the effects of calcium ions, factor XIIIa activity, and different thrombin concentrations and on the rate and degree of clot contraction.

The new technique to quantify the kinetics of blood clot contraction is based on the continuous tracking of dynamics of clot size by sensing changes in the light scattering of the clot over time followed by computational processing of the images. The optical platform for this method is the commercially available Thrombodynamics Analyser System (HemaCore, Moscow, Russia) initially designed to monitor the directional growth of a plasma clot triggered on one side of a cuvette with immobilized tissue factor. A standard procedure to analyze clot contraction with this instrument is described next. Plastic cuvettes 12 x 7 x 1 mm are pre-lubricated with a detergent to prevent attachment of fibrin to the walls, which precludes clot contraction. Fresh citrated human blood is incubated with calcium chloride (2 mM final concentration) at 37°C for 3 minutes followed by addition of 1 U/ml of freshly thawed thrombin to initiate blood clotting and platelet activation. Thrombin-activated whole blood sample (80 µl) is quickly transferred to the cuvette, which is placed into the thermostatic chamber (37°C) between a red LED and an optical sensor (CCD camera). The clot contraction is monitored by taking images every 15 seconds over 20 minutes or more. Off-line automated computational analysis of the serial images provides a kinetic curve plotted as the degree of contraction (% retracted clot volume) as a function of time. This curve is characterized by the following parameters: lag period (min), average contraction velocity (Δ clot volume/time), final degree of contraction (%), time to reach 1/4 and 1/2 of the final contraction (min), and area under the curve (a.u.).

A general conclusion based on the examination of about 100 kinetic curves is that clot contraction is a non-monotonic process and has at least two phases characterized by distinct rates. Variation of [Ca²⁺] from zero up to 10 mM revealed that exogenous Ca²⁺ was not indispensable for clot contraction to occur; however, the clots formed without addition of Ca²⁺ were less stable and often extruded red blood cells from the clot during contraction (likely due to impaired fibrin cross-linking with factor XIIIa). Recalcification of blood with 2 or 5 mM Ca²⁺ stabilized the clot, preventing the red blood cell fallout, but had no significant effect on the contraction kinetics. When added at a final concentration of 10 mM, Ca²⁺ partially inhibited clot contraction as confirmed by significantly reduced average velocity and the degree of retraction compared to the Ca²⁺-free conditions, perhaps due to suppression of platelet function. These findings indicate that 2 to 5 mM [Ca²⁺] is optimal for contraction of a whole blood clot in vitro. To see if clot contraction and stability depends on the cross-linking catalyzed by factor XIIIa, we studied clot contraction in the presence of 1 mM iodoacetamide, a potent inhibitor of the transglutaminase activity. Iodoacetamide did not affect blood clot formation but abolished clot shrinkage, confirming that the enzymatic activity of factor XIIIa is essential for clot contraction. As expected, blood clot contraction depended on thrombin concentration within a 0.1-1.0 U/ml range. Thrombin enhanced the rate and degree of clot contraction in a dose-dependent manner.

In summary, we developed an accurate and simple assay for blood clot contraction which can be used for research and may be potentially useful for in vitro diagnostics.

(Research supported by the Program of Competitive Growth of Kazan Federal University)