Contact activation of blood coagulation on a defined kaolin/collagen surface in a microfluidic assay

Abstract

Generation of active Factor XII (FXIIa) triggers blood clotting on artificial surfaces and may also enhance intravascular thrombosis. We developed a patterned kaolin (0 to 0.3 pg/μm2)/type 1 collagen fibril surface for controlled microfluidic clotting assays. Perfusion of whole blood (treated only with a low level of 4 μg/mL of the XIIa inhibitor, corn trypsin inhibitor) drove platelet deposition followed by fibrin formation. At venous wall shear rate (100 s−1), kaolin accelerated onset of fibrin formation by ~100 sec when compared to collagen alone (250 sec vs. 350 sec), with little effect on platelet deposition. Even with kaolin present, arterial wall shear rate (1000 s−1) delayed and suppressed fibrin formation compared to venous wall shear rate. A comparison of surfaces for extrinsic activation (tissue factor TF/collagen) versus contact activation (kaolin/collagen) that each generated equal platelet deposition at 100 s−1 revealed: (1) TF surfaces promoted much faster fibrin onset (at 100 sec) and more endpoint fibrin at 600 sec at either 100 s−1 or 1000 s−1, and (2) kaolin and TF surfaces had a similar sensitivity for reduced fibrin deposition at 1000 s−1 (compared to fibrin formed at 100 s−1) despite differing coagulation triggers. Anti-platelet drugs inhibiting P2Y1, P2Y12, cyclooxygenase-1 or activating IP-receptor or guanylate cyclase reduced platelet and fibrin deposition on kaolin/collagen. Since FXIIa or FXIa inhibition may offer safe antithrombotic therapy, especially for biomaterial thrombosis, these defined collagen/kaolin surfaces may prove useful in drug screening tests or in clinical diagnostic assays of blood under flow conditions.

FXIa and platelet polyphosphate as therapeutic targets during human blood clotting on collagen/tissue factor surfaces under flow

“…32 We used a microfluidic assay of platelet deposition and coagulation on a type I fibrillar collagen/lipidated tissue factor (TF) surface that could trigger thrombin generation via the contact pathway and/or the extrinsic pathway. 24,33 We used two distinct monoclonal anti-FXI antibodies, 14E11 and O1A6, to explore the role of FXI in contact activation and in promoting thrombin amplification. 14E11 selectively inhibits FXI activation by FXIIa but not FXI activation by thrombin, and O1A6 interferes with both FXI activation by FXIIa and factor IX (FIX) and FV activation by FXIa.…”

“…With 4 mg/mL CTI, drawn blood does not clot in the reservoir during the experiment but will generate thrombin via FXIIa in ;300 seconds when perfused over collagen or surface-linked contact activators. 33 We investigated the role of platelet-derived polyP in promoting thrombin generation and enhancing fibrin structure and clot stability by inhibiting polyP with polyP-binding protein (PPXbd), the recombinant polyPbinding domain of E coli exopolyphosphatase (concentration that inhibits 50%, 8.5 mg/mL). 17,22 This microfluidic data with human blood demonstrated specific conditions during which FXIa and platelet polyP play a kinetically significant role in clotting under flow conditions that can be targeted with inhibitors.…”

Dynamics of Thrombin Generation and Flux from Clots during Whole Human Blood Flow over Collagen/Tissue Factor Surfaces

“…By recreating hemodynamic flow over procoagulant surfaces, the dynamics and pharmacology of thrombin generation can be studied with human blood ex vivo. In microfluidic clotting assays, fibrin generation is often used as an indirect indicator for thrombin activity (5,(37)(38)(39)(40). Our laboratory previously developed a peptide-based platelet targeting biosensor to report platelet associated thrombin activity in both microfluidic and animal thrombosis models (23,24).…”

In microfluidico: Recreating in vivo hemodynamics using miniaturized devices

“…Numerous inhibitors and experimental designs allow control of these pathways to obtain clotting conditions that range from the contact pathway dominated to the extrinsic pathway dominated (Fig. 1(B)) [10,11]. …”