In vivo and ex vivo impact of THBD and PROCR polymorphisms on the anticoagulant response of the protein C pathway.

Inhibitionn of the tissue factor pathway of coagulation by recombinantt nematode anticoagulant protein c2 in patientss undergoing elective coronary stent implantation: effectss on hemostatic and inflammatory parameters

Thrombomodulin Gly430Ser mutation attenuates the affinity with thrombin and contributes to thrombosis development.

Thrombin dynamics and clinical events in cirrhosis: low prothrombin conversion predicts bleeding risk

Pro- and anti-coagulant factors are significantly altered in liver cirrhosis resulting in an increased bleeding and thrombosis risk. The thrombin generation (TG) test often seems unaltered in cirrhosis, indicating that a new hemostatic balance is reached. TG is determined by prothrombin conversion and thrombin inactivation. These processes can be quantified using the computational thrombin dynamics (TD) add-on analysis for the TG test. We studied TD parameters in cirrhosis to explore its predictive potential for bleeding and thrombosis. In the current study, 32 CP-A, 22 CP-B and 41 CP-C cirrhosis patients and 20 healthy controls were enrolled. Cirrhosis was predominantly alcohol-related (52%), while viral and metabolic etiologies accounted for 34% and 14% of cases, respectively. Patients receiving anticoagulant therapy were excluded. TG was measured using PPP Reagent Low in the presence and absence of thrombomodulin. TG peak height and velocity index were higher in patients than controls (+20%,p = 0.039 and +70%,p = 0.002), and time-to-peak was shorter (−18%,p < 0.0001). TD parameters were obtained computationally based on TG and coagulation factor data. The amount of prothrombin converted (PCtot) was significantly reduced in patients (−43%,p < 0.0001), and the magnitude of the reduction was associated with disease severity. The maximum rate of prothrombin conversion (PCmax) showed a trend towards higher levels in CP-A patients. On the anticoagulant slide, the thrombin decay capacity (TDC) was significantly lower in patients (−44%,p < 0.0001), indicating a newly developed balance between pro- and anticoagulant processes that result in a slightly procoagulant TG phenotype. Four patients experienced bleeding and seven experienced thrombosis during follow-up (median = 499 days; CI: 478–524 days). Low maximum prothrombin conversion rates (PCmax) were associated with an increased hazard of bleeding. Patients in the lowest 20% of PCmax had a HR of 12.9 (95% CI 1.34–124.31, p = 0.027) for bleeding. Low thrombin-α2Macroglobulin (T-α2M) complex formation was significantly associated with thrombosis during follow-up. Patients in the lowest quintile of T-α2M had a HR of 6.6 (95% CI 1.47–29.9, p = 0.014) for thrombosis. We conclude that reduced prothrombin conversion and thrombin inactivation result in rebalanced TG in cirrhosis patients. Our results demonstrate the potential clinical usefulness of TD parameters for the stratification of bleeding and thrombotic risk in cirrhosis patients.

The Tissue Factor-TFPI Pathway as a Therapeutic Target in Bleeding Disorders of Unknown Cause.

BACKGROUND Bleeding disorders of unknown cause (BDUC) comprise a heterogeneous group of conditions characterized by clinically significant bleeding despite normal results on standard haemostatic investigations. OBJECTIVES To investigate tissue factor (TF)-dependent coagulation in patients with BDUC and to evaluate the in vitro ability of concizumab and recombinant activated factor VII (rFVIIa) to restore thrombin generation. METHODS AND RESULTS Thrombin generation was assessed using an assay designed to isolate TF-dependent coagulation by inhibiting contact pathway activation with corn trypsin inhibitor (CTI). A subgroup of patients with BDUC exhibited impaired thrombin generation under these conditions. Reduced thrombin generation was frequently associated with increased tissue factor pathway inhibitor (TFPI) activity, suggesting that excessive inhibition of TF-mediated coagulation contributes to the bleeding phenotype in a subset of patients. In vitro addition of concizumab or rFVIIa restored thrombin generation, indicating that defects in TF-dependent coagulation can be pharmacologically corrected. CONCLUSIONS These findings identify dysregulation of the TF-TFPI axis as a potential pathophysiological mechanism underlying bleeding in a subset of patients with BDUC and highlight TFPI inhibition and rFVIIa administration as potential therapeutic approaches. However, the absence of abnormalities in all patients supports the concept that BDUC represents a biologically heterogeneous group of disorders with multiple underlying mechanisms.

Correlation Between Von Willebrand Factor, Thrombin-Antithrombin Complex, and Disease Severity in Sickle Cell Disease: Implications for Thrombosis

Introduction Sickle cell disease (SCD) is a hypercoagulable state characterized by elevated procoagulant factors, including Von Willebrand factor (VWF), and enhanced thrombin generation, marked by increased thrombin–antithrombin complex (TAT). Research on VWF and TAT activity during vaso-occlusive crises (VOC) remains scarce in our region. Objective To determine VWF antigen and TAT complex serum levels in SCD during steady state and VOC, and correlate these with disease severity. Methodology This cross-sectional study evaluated 30 SCD patients during VOC and re-evaluated the same cohort during their steady state. A control group of 30 HbAA individuals was included. Serum was analyzed for VWF and TAT complex. Analysis of Variance (ANOVA) and Pearson correlation tests were utilized (p < 0.05). Results Mean VWF levels were highest during VOC (500.9 ± 175.6 ng/mL), followed by steady state (421.5 ± 130.9 ng/mL) and controls (395.0 ± 115.2 ng/mL) (p=0.014). TAT complex levels were highest during VOC (28.9 ± 10.4 ng/mL) compared to steady state (24.1 ± 7.8 ng/mL) and controls (4.0 ± 4.2 ng/mL) (p<0.001). Steady-state VWF positively correlated with TAT (r = 0.458, p = 0.011), while steady-state TAT positively correlated with disease severity (r = 0.401, p = 0.028). Conclusion VWF and TAT levels are significantly elevated in SCD patients, with further accentuation during VOC. These elevations strongly correlate with disease severity and specific clinical complications, establishing them as vital markers for evaluating thrombotic risk in SCD patients.

Shaping the thrombotic cascade

SUMMARY Venous thromboembolism is a common thrombotic disorder presenting complex clinical challenges, particularly regarding the optimal duration of anticoagulation therapy. Extending anticoagulation reduces the risk of recurrence but increases bleeding risk. Accurate prediction of recurrence risk could help guide decisions on whether to continue or stop anticoagulation. In Chapter 2, we investigated platelet and endothelial biomarkers and found that elevated levels of von Willebrand Factor during anticoagulation were associated with early recurrence, especially in men. These findings suggest VWF might be a useful biomarker for personalized risk prediction, enabling more tailored anticoagulation strategies if validated in further studies. Chapter 3 employed unbiased mass spectrometry to discover new biomarkers but did not find proteins that significantly differed between patients with and without recurrence, either during or after anticoagulation. Chapter 4 evaluated global coagulation assays focusing on thrombin generation and thrombin dynamics. We found that reduced endogenous thrombin potential (ETP) and reduced thrombin inhibition by antithrombin were linked to recurrence, particularly when excluding hormone-related VTE cases. Additionally, impaired thrombomodulin-mediated inhibition of thrombin generation was associated with recurrence risk. Chapter 5 shifted focus to patients with immune thrombocytopenia (ITP) , a patient group with heightened thrombotic risk. These patients showed elevated ETP, which increased further following eltrombopag treatment, highlighting a prothrombotic state. After establishing the importance of thrombin generation in predicting recurrence and assessing thrombotic state, Part II of the thesis examined thrombomodulin’s ability to modulate thrombin generation for diagnostic and therapeutic purposes, focusing on strategies to enhance or inhibit its activity. In Chapter 6, we developed liposomes conjugated with thrombomodulin (TM-liposomes) that more effectively inhibited thrombin generation in plasma and whole blood compared to soluble thrombomodulin. The lipid composition of these liposomes influenced their efficacy, with higher phosphatidylserine content enhancing protein C activation. These TM-liposomes may improve diagnostics by more closely mimicking the natural endothelial membrane environment and enabling better assessment of the activated protein C pathway in thrombin generation. Chapter 7 describes the design of a nanobody (VhH) that inhibits thrombin-activatable fibrinolysis inhibitor (TAFI) activation by thrombomodulin. We demonstrated that this VhH enhanced fibrinolysis in whole blood under flow conditions, offering a new strategy to enhance clot breakdown and possibly reduce thrombotic complications.