Platelet-primed interactions of coagulation and anticoagulation pathways in flow-dependent thrombus formation

Abstract

In haemostasis and thrombosis, platelet, coagulation and anticoagulation pathways act together to produce fibrin-containing thrombi. We developed a microspot-based technique, in which we assessed platelet adhesion, platelet activation, thrombus structure and fibrin clot formation in real time using flowing whole blood. Microspots were made from distinct platelet-adhesive surfaces in the absence or presence of tissue factor, thrombomodulin or activated protein C. Kinetics of platelet activation, thrombus structure and fibrin formation were assessed by fluorescence microscopy. This work revealed: (1) a priming role of platelet adhesion in thrombus contraction and subsequent fibrin formation; (2) a surface-independent role of tissue factor, independent of the shear rate; (3) a mechanism of tissue factor-enhanced activation of the intrinsic coagulation pathway; (4) a local, suppressive role of the anticoagulant thrombomodulin/protein C pathway under flow. Multiparameter analysis using blood samples from patients with (anti)coagulation disorders indicated characteristic defects in thrombus formation, in cases of factor V, XI or XII deficiency; and in contrast, thrombogenic effects in patients with factor V-Leiden. Taken together, this integrative phenotyping approach of platelet-fibrin thrombus formation has revealed interaction mechanisms of platelet-primed key haemostatic pathways with alterations in patients with (anti)coagulation defects. It can help as an important functional add-on whole-blood phenotyping. In the processes of haemostasis and thrombosis, platelet and coagulation pathways are tightly linked, with platelets both responding to thrombin and fibrin and, conversely, providing a phosphatidylserine-exposing surface on which high levels of thrombin and fibrin are formed 1-4. Thrombotic and bleeding disorders are mostly related to a dysregulation of one of these pathways, for instance when linked to platelet or coagulation factor mutations, or to the (combined) use of antiplatelet or anticoagulant medication. In the last decade, it has become clear that the accumulation of a platelet-and fibrin-containing plug or thrombus is instrumental to the onset of haemostatic as well as thrombotic events 5-7. An important modifying factor in thrombus development is provided by the local blood flow 8,9. This implies that, for adequate and comprehensive monitoring of the thrombotic process, measurements are to be performed under conditions of flow, preferentially recording both platelet and coagulation activation at the same time.

Inhibition of platelet adhesion, thrombus formation, and fibrin formation by a potent αIIbβ3 integrin inhibitor from ticks

“…Clean and degreased coverslips were coated with 2 microspots (5 mm center‐to‐center distance; 1 µL of 50 µg/mL collagen‐I). After 1 hour of incubation and a washing step with saline, the downstream microspot was co‐coated with TF (1 µL of 500 pM), similarly as described elsewhere 27 . Before the experiment, citrated‐anticoagulated blood samples were preincubated with disagregin (100 nM, 1 µM), RGD‐disagregin (100 nM, 1 µM) or eptifibatide (1 µM) for 5 minutes.…”

“…Using the scaled values, subtraction was conducted to visualize the difference between control and treatment. In heat maps with fibrin formation, times to first fibrin formation were subtracted from 14 minutes 27 …”

“…28 The following output parameters were used (Table S3) 1, multiple single adhered platelets; 2, platelet monolayer; 3, small aggregates; 4, medium-size aggregates; and 5, large aggregates. [26][27][28][29] Thrombus multilayer (P5) and thrombus contraction (P6) were scored from 0 to 3. 28 The following fibrin parameters were used: fibrinogen or fibrin deposition (P7), fibrin formation (P8) scored from 0 to 3.…”

Platelet Membrane Receptor Proteolysis: Implications for Platelet Function

“…Factor Xa itself is generated (from non-cleaved factor X) at the surface of procoagulant, phosphatidylserine-exposing platelets, where it serves to produce thrombin ( 42 , 43 ). The combined activities of factor Xa and thrombin can be measured in thrombin generation assays under stasis and are also detectable under flow conditions in whole blood assays ( 12 , 44 ). Intriguingly, the factor Xa receptor PAR2, present on endothelial cells ( 45 ), does not play a significant role in human platelet activation ( 46 ).…”

Table_1.XLSX

“…Factor Xa itself is generated (from non-cleaved factor X) at the surface of procoagulant, phosphatidylserine-exposing platelets, where it serves to produce thrombin (42,43). The combined activities of factor Xa and thrombin can be measured in thrombin generation assays under stasis and are also detectable under flow conditions in whole blood assays (12,44). Intriguingly, the factor Xa receptor PAR2, present on endothelial cells (45), does not play a significant role in human platelet activation (46).…”