A new method utilizing image capture and processing was developed for the analysis of blood plasma coagulation at surfaces. The coagulation was detected in a cuvette by time-lapse image capture of light scattering from the developing fibrin network. By image processing and computer analysis of the captured image data, both early detection of coagulation at the surface and the propagation phase of coagulation could be measured in the same experiment. It is possible to use both platelet-rich plasma (PRP) and platelet-free plasma (PFP) with the method, and thereby study the platelet contribution to both surface coagulation and propagation of coagulation. Two well-known model surfaces, hydrophilic and hydrophobic glass, were used in combination with PRP and PFP to illustrate the method. Hydrophilic glass activated coagulation significantly faster (PRP: 7.0 +/- 1.7 min, PFP: 5.9 +/- 1.2 min, n= 16) than hydrophobic glass (PRP: 50 +/- 14 min, PFP: 65 +/- 32 min, n = 16) in both PRP and PFP. Hydrophilic surfaces showed a faster initial propagation of coagulation adjacent to the surface (mean velocity: 0.14 +/- 0.05 mm/ minute) compared with the propagation observed further out from the surface (mean velocity: 0.05 +/- 0.01 mm/min). The method is very flexible and can be suitable for screening hemocompatibility of biomaterials.

In the present study the initial blood compatibility of glycerol monooleate (GMO)-coated surfaces was evaluated after deposition to surfaces and in bulk. The model surface was silica onto which multiple layers of fibrinogen or human serum albumin (HSA) was immobilized. The protein-coated surfaces were subsequently dip-coated in GMO in ethanol and used for blood plasma and whole blood experiments. The characterization methods included null ellipsometry, scanning electron microscopy, imaging of coagulation, hemolysis test and whole blood coagulation time by free oscillation rheometry.

The results showed a GMO film thickness of approximately 350 angstrom (similar to 4 mu g/cm(2)) upon dip-coating in ethanolic solution. A major part of the deposited layer detached in aqueous solutions, especially during shear conditions. The coagulation time on GMO was significantly prolonged compared to that on HSA coated silica. Whole blood tests showed that GMO is a very weak hemolytic agent. Deposited GMO detached easily from surfaces upon rinsing or shearing, although a stable layer with undefined phase structure and a thickness of 50-70 angstrom remained on HSA and fibrinogen precoated surfaces. This indicates that GMO has stronger adhesive forces to its substrate compared to the cohesive forces acting within the bulk GMO. The ability of GMO to detach from itself and tentatively form micelles or lipid bilayers when subjected to flowing blood may be of use in extravascular applications. It is concluded that GMO results in weak blood activation, and the material may in spite of this be suitable in selected biomaterial applications, especially as a biosealant and in colloidal dispersions.

In this work we have evaluated the haemocompatibility of different surface modifications, intended for biomaterials and biosensor applications. Polystyrene slides were coated with thin hydrogel films by self-initiated photografting of four different monomers. The hydrogel surface modifications were thoroughly characterized and tested for their protein resistance and ability to facilitate platelet adhesion and activation of the coagulation system. There was very little protein adsorption from human plasma on the hydrogels formed from poly(ethylene glycol) methacrylate (PEGMA) and 2-hydroxyethyl methacrylate (HEMA). Platelet adhesion tests performed under both static and flow conditions showed that these coatings also demonstrated very high resistance towards platelet adhesion. A small amount of platelets were found to adhere to hydrogels formed from ethylene glycol methyl ether methacrylate (EGMEMA) and 2-carboxyethyl methacrylate (CEA). The polystyrene substrates themselves facilitated large amounts of platelet adhesion under both static and flow conditions. Utilizing a novel setup for imaging of coagulation, it was shown that none of the hydrogel surfaces activated the coagulation system to any great extent. We suggest that this simple fabrication method can be used to produce hydrogel coatings with unusually high blood compatibility, suitable for demanding biomaterials applications.

The purpose of this work was to investigate the relationship between surface charge of phospholipid membranes and coagulation. Also, we wanted to demonstrate that coagulation at phospholipid membranes could successfully be studied in the method for imaging of coagulation.

Analytical procedure: Supported phospholipid membranes were formed from palmitoyl-oleoyl-glycero-3-ethylphosphocholine (POEPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1- palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine] (POPS) on silicon substrates. The surface charge of the phospholipid membranes was controlled by using different compositions of POPS (negative net charge), POPC (weak negative net charge) and POEPC (positive net charge). Imaging of coagulation experiments were performed on all phospholipid membrane coated surfaces as well as the clean silicon substrate. The experiments were performed in platelet-free plasma (PFP) diluted 50:50 with phosphate-buffered saline (PBS).

Results: Comparing the negatively charged SiO₂ surface with the negatively charged POPS (30%)/POPC(70%) we found an interesting difference. Although both surfaces activated coagulation rapidly, the POPS surface facilitated a faster propagation of coagulation from the surface than the SiO2 surface. It was also found that in order for the phospholipid membranes to exert procoagulant properties, the POPS content in the membrane had to exceed ~6 %. It was also found that positively charged phospholipid membranes did not induce activation of coagulation.

Conclusions: The work in this paper demonstrated that the coagulation process at phospholipid membranes can be studied in a straightforward manner using the imaging of coagulation setup. Furthermore, we speculate that the negatively charged phospholipid membranes but not the SiO2 surface can support the binding of coagulation factor complexes, thus facilitating a faster propagation of coagulation. The fact that the POPS content must exceed ~ 6% to fully exert procoagulant properties was also a very interesting result, especially since platelets, when activated, become procoagulant by increasing their negatively charged phosphatidylserine exposure from ~0 % to maximally ~10 %.

The physiological relevance and function of coagulation factor XII (FXII), the first zymogen in the intrinsic pathway, has for a long time been a matter of debate. The aim of this study was to shed some light on the role of factor XII in thrombus formation with a focus on its effect during the propagation phase of coagulation. In order to study propagation of coagulation we utilized a new imaging method to measure propagation rates from an activating surface in both platelet-free plasma and platelet-rich plasma. The most essential results revealed that both FXII and its substrate FXI are located on the surface of activated platelets. The surface of preexisting clots does not support coagulation in a FXII dependent manner. However, we found strong evidence for an accelerated propagation of tissue factor initiated coagulation when contact activation of FXII simultaneously occurred in the proximity. In vivo sources for contact activation may be exposed subendothelial collagen as well as soluble and cell derived poly-anions. If such in vivo contact activation of FXII occurs, even though moderate, it could contribute to in vivo thrombus growth rate and thus be of pathophysiological importance.