Increased microvesicle-associated thrombin generation in patients with immune thrombocytopenia after initiation of thrombopoietin receptor agonists

Immune thrombocytopenia (ITP) patients have thrombocytopenia and increased bleeding risk, but, conversely, they also have increased thrombotic risk which appears to be exacerbated by thrombopoietin-receptor agonist (TPO-RA)-treatment. Microvesicles (MVs) released from activated/apoptotic cells are prothrombotic due to exposure of phosphatidylserine (PS) and tissue factor (TF). MVs are increased in ITP patients, but their prothrombotic effect, before and during treatment with TPO-RAs, is unclear. We studied the effect of TPO-RAs on the procoagulant activity of MVs in 11 ITP patients, before, and two and six weeks after initiation of treatment, and in 15 healthy controls. MV-associated PS-activity, TF-activity and the capacity of isolated MVs and plasma to generate thrombin in a phospholipid-dependent manner were measured. Before treatment with TPO-RAs, prothrombotic markers in ITP patients were comparable to levels found in healthy controls. After both two and six weeks of TPO-RA-treatment, ITP patients had higher MV-associated PS-activity and phospholipid-dependent thrombin generation in plasma than controls. In addition, ITP patients had increased phospholipid-dependent MV-associated thrombin generation two weeks after initiation of TPO-RA-treatment compared with controls and pre-treatment levels. MV-associated TF-activity was low in controls and in ITP patients before and after initiation of TPO-RA-treatment. In conclusion, TPO-RAs increase phospholipid-dependent MV-associated thrombin generation in ITP patients. This could contribute to or exacerbate a pre-existing hypercoagulable state. Phospholipid-dependent thrombin generation generated by isolated MVs, or measured directly in plasma, may be potential tools that could help in the risk-assessment of future thromboembolic events in ITP patients, both before and after initiation of TPO-RA-treatment

The reversal effect of prothrombin complex concentrate (PCC), activated PCC and recombinant activated factor VII against anticoagulation of Xa inhibitor

Background An increasing number of patients are treated with direct-acting oral anticoagulants (DOACs), but the optimal way to reverse the anticoagulant effect is not known. Specific antidotes are not available and prothrombin complex concentrate (PCC), activated PCC (aPCC) and recombinant factor VIIa (rFVIIa) are variously used as reversal agents in case of a major bleeding. We aimed to determine the most effective haemostatic agent and dose to reverse the effect of rivaroxaban in blood samples from patients taking rivaroxaban for therapeutic reasons. Methods Blood samples from rivaroxaban-treated patients (n = 50) were spiked with PCC, aPCC and rFVIIa at concentrations imitating 80%, 100% and 125% of suggested therapeutic doses. The reversal effect was assessed by thromboelastometry in whole blood and a thrombin generation assay (TGA) in platelet-poor plasma. Samples from healthy subjects (n = 40) were included as controls. Results In thromboelastometry measurements, aPCC and rFVIIa had a superior effect to PCC in reversing the rivaroxaban-induced lenghtening of clotting time (CT). aPCC was the only haemostatic agent that shortened the CT down to below the control level. Compared to healthy controls, patients on rivaroxaban also had a prolonged lag time and decreased peak concentration, velocity index and endogenous thrombin potential (ETP) in platelet-poor plasma. aPCC reversed these parameters more effectively than rFVIIa and PCC. There were no differences in efficacy between 80%, 100% and 125% doses of aPCC. Conclusions aPCC seems to reverse the anticoagulant effect of rivaroxaban more effectively than rFVIIa and PCC by evaluation with thromboelastometry and TGA in vitro

MV-associated thrombin generation in pooled normal plasma (PNP) without corn trypsin inhibitor (CTI).

<p>MVs were isolated from plasma, washed (17,000 g, 30 min, RT) and added to PNP without CTI. Thrombin generation was measured -/+ MP-reagent (phospholipids) and -/+ anti-TFPI antibodies (abs). A) Thrombin generation curves of MVs from pancreatic cancer patients (black) and healthy controls (green). B) LT, ETP, peak and velocity index of MVs from pancreatic cancer patients (filled circles) and healthy controls (open circles) in PNP with anti-TFPI abs added. Lines indicate mean values, and * indicates significant differences between the pancreatic cancer group and the healthy control group (RM two-way ANOVA with Sidak’s multiple comparison test, adjusted p-values are shown).</p

The effect of inhibiting antibodies against tissue factor (TF) on microvesicle (MV)-associated thrombin generation in pooled normal plasma (PNP) with corn trypsin inhibitor (CTI).

<p>The thrombin generation (lag time) of MVs isolated from pancreatic cancer patients plasma (A) or of MVs isolated from plasma obtained from citrated whole blood that had been incubated with <i>Neisseria meningitidis</i> (10⁸/mL) (B). MVs were analyzed with (circles) or without (squares) preincubation with anti-TF abs, in the presence or absence of phospholipids (MP-reagent) and antibodies against TFPI (anti-TFPI abs). The patient sample with the shortest lag time and the most evident TF-dependent thrombin generation across the four conditions is marked in red.</p

Microvesicle (MV)-associated thrombin generation in pooled normal plasma (PNP) with corn trypsin inhibitor (CTI).

<p>MVs were isolated from plasma, washed (17,000 g, 30 min, RT) and added to CTI-containing PNP. Thrombin generation was measured -/+ MP-reagent (phospholipids) and -/+ anti-TFPI antibodies (abs). A) Thrombin generation curves of MVs from pancreatic cancer patients (black) and healthy controls (green). B) Lag time (LT) of MVs from pancreatic cancer patients (filled circles) and healthy controls (open circles). Lines indicate mean LT, and * indicates significant differences (RM two-way ANOVA with Sidak’s multiple comparison test, adjusted p-values are shown).</p