Role of Tissue Factor in Platelet Adhesion: Real-Time Perfusion Studies.

Exposure of Tissue Factor (TF) to circulating blood and excessive thrombin generation are major contributors to acute thrombosis. Platelets also play a central role in the pathogenesis of occlusive events. Despite the importance of both components, convincing evidence of direct adherence of platelets onto TF immobilized on a surface is lacking. Here, we studied the interactions of platelets with different TF-rich substrata. Real-time perfusion techniques were applied to visualize thrombus formation and to evaluate the time necessary for platelets to both attach to the surface and to form thrombi. We used three different sources of TF as substrates:

• Thromborel S^®, which is TF purified from human placenta (pTF) (at 0.4 ng/cm²);

• Innovin^®, recombinant TF that has been relipidated (rTF) (at 0.4 ng/cm²); and

• TF expressed on the membranes of TF-transfected CHO cells (CHO-TF).

As negative controls, we used equivalent surfaces sprayed with 0.5% bovine serum albumin or cultured sham-transfected CHO cells (CHO-C-), respectively. Platelet-rich plasma (PRP) from blood anticoagulated with low-molecular-weight heparin was perfused over these substrata at a shear rate of 300s⁻¹ for 5 min. Images were captured with a digital camera at high resolution and streamed to a computer. Continuous real-time photographs were taken during the full 5 min. Single frames were captured at predetermined times and the mean area of the platelet aggregates at these times was measured. Every experiment was replicated at least 4 times. Results are expressed as mean ± S.E.M. Under these conditions, platelets bound and formed thrombi on all substrata containing TF, although the time to aggregate formation and aggregate area varied depending on the substratum. Platelet deposition was minimal on the BSA-coated surfaces or on CHO-C- cells.

Initial platelet deposits on pTF were observed after 120 sec of perfusion. After 5 min, thrombi reached an area of 122±41 μm². Relipidated rTF was less effective in supporting platelet deposition, requiring 135 seconds on average for the initiation of platelet aggregates. After 5 min, the average area of platelet aggregates was 34.3±9.6 μm². Formation of platelet aggregates on CHO-TF cells was noticeable after 80 seconds. Aggregates formed at 5 min reached an area of 213±58 μm². In conclusion, our studies demonstrate that platelets adhere to and aggregate on different TF substrata. The presence of other adhesive molecules in cell-derived TF preparations (pTF, CHO-TF) appears to accelerate the interaction of platelets with TF. These observations suggest that in addition to its role in blood coagulation, TF also has role in platelet adhesion by providing an adhesive surface at sites of vessel injury.

SAF2003-05780, SGR2005-00952, FIS PI040887, FIS CP04-0011, NHLBI 5R01HL064796.