Protocol for the Assessment of Endogenous Platelet-Associated Tissue Factor-Dependent Thrombin Generation in Human...

Platelet-associated Tissue Factor (TF) is increasingly recognized as a functionally active determinant of platelet procoagulant activity, enabling platelets to initiate and sustain thrombin generation beyond their established role as a phospholipid surface for coagulation factor assembly. However, its specific functional assessment remains challenging, as conventional Calibrated Automated Thrombogram (CAT) assays rely on exogenous TF to trigger thrombin generation, thereby masking the endogenous contribution of platelet-associated TF. Here, we describe a Calibrated Automated Thrombogram (CAT)-based workflow that enables the selective assessment of TF-dependent thrombin generation in isolated intact human platelets under conditions that preserve the contribution of endogenous platelet-associated TF. The protocol integrates key methodological steps, including inhibition of contact pathway activation through the use of microvesicle-free plasma pool containing a contact pathway inhibitor, normalization of the procoagulant membrane surface by exogenous phospholipids, and functional validation through neutralizing antibodies against TF and Tissue Factor Pathway Inhibitor (TFPI). The workflow also addresses key pre-analytical variables related to blood collection and platelet-rich plasma (PRP) preparation. Under these controlled conditions, thrombin generation kinetics -particularly LagTime- reflect the functional activity of platelet-associated TF, as demonstrated by their modulation upon TF and TFPI inhibition and their association with TF^pos-platelets levels measured by flow cytometry. By avoiding the addition of exogenous TF, this approach overcomes key methodological limitations that have contributed to the ongoing controversy surrounding platelet-associated TF, providing a functional readout that complements antigen-based measurements. This protocol therefore represents a robust and reproducible framework for investigating platelet-associated TF in both mechanistic and clinical studies.