Determination of surface tissue factor thresholds that trigger coagulation at venous and arterial shear rates: amplification of 100 fM circulating tissue factor requires flow.

Protein microarrays presenting spots of collagen and lipidated tissue factor (TF) allowed a determination of the critical surface concentration of TF required to trigger coagulation under flow. Whole blood supplemented with corn trypsin inhibitor (to inhibit factor XIIa) was perfused over microarrays for 5 minutes. Immunofluorescence staining of platelet glycoprotein GPIbalpha and fibrin(ogen) revealed a critical TF concentration (EC50) of 3.6, 8.4, and 10.2 molecules-TF/microm2 at wall shear rates of 100, 500, and 1000 s(-1), respectively. For collagen arrays where only the center lane of spots (in the direction of flow) contained TF, a downstream distance of 14 mm was required for the thrombus to widen enough to reach across a 300-micrometer gap to the adjacent TF-free lanes of collagen spots, in agreement with numerical simulation. To investigate the effect of low levels of circulating TF, whole blood (+/-100 fM added TF) was tested under static and flow conditions. After 5 minutes, the addition of 100 fM TF to whole blood had negligible effect under static conditions, but caused a 2.5-fold increase in fibrin formation under flow. This report defines the threshold concentrations of surface TF required to trigger coagulation under flow.

Matrix protein microarrays for spatially and compositionally controlled microspot thrombosis under laminar flow.

Microarraying allows the spatial and compositional control of surfaces, typically for the purpose of binding reactions. Collagen and/or von Willebrand Factor (vWF) in 5% glycerol was contact printed onto glass slides to create defined microspots (176-microm diameter) of adsorbed protein without sample dehydration. The arrays were mounted on flow chambers allowing video microscopy during perfusion (wall shear rate of 100-500 s(-1)) of recalcified corn trypsin inhibitor-treated whole blood or platelet rich plasma and subsequent array scanning via anti-GPIbalpha and anti-fibrin(ogen) immunofluorescence. To mimic the subendothelial matrix, vWF was microarrayed over sonicated type I collagen microspots. For whole blood perfusion (500 s(-1), 10 min) over collagen, vWF, and collagen/vWF microspots, the amount of platelet deposition on the collagen/vWF spots was approximately 2 times greater in comparison to the collagen spots and approximately 18 times greater in comparison to the vWF spots. The amount of fibrin(ogen) deposition on the collagen/vWF spots was approximately 2 times greater in comparison to the collagen spots and approximately 4 times greater in comparison to the vWF spots. This protocol allowed for highly uniform (CV = 18%) and precisely located thrombus formation at a density of >or=400 spots/cm(2). Microarrays are ideal for the combinatorial assembly of adhesive and procoagulant proteins to study thrombosis as well as to study axial and lateral transport effects between discrete microspots of distinct composition.