Coenraad Hemker

Preoperative thrombin generation is predictive for the risk of blood loss after cardiac surgery: a research article

BackgroundIn this study the value of thrombin generation parameters measured by the Calibrated Automated Thrombography for prediction of blood loss after cardiac surgery with cardiopulmonary bypass was investigated.MethodsThirty male patients undergoing first-time coronary artery bypass grafting were enrolled. Blood samples were taken pre-bypass before heparinisation (T1) and 5 min after protamine administration (T2). Thrombin generation was measured both in platelet-rich plasma and in platelet-poor plasma. Besides thrombin generation measurements, activated clotting time, haematocrit, haemoglobin, platelet number, fibrinogen, antithrombin, D-dimers, prothrombin time and activated partial thromboplastin time were determined. Blood loss was measured and the amount of transfusion products was recorded postoperatively until 20 hours after surgery. Patients were divided into two groups based on the median volume of postoperative blood loss (group 1: patients with median blood loss <930 ml; group 2: patients with median blood loss ≥930 ml).ResultsOn T1, patients of group 2 had a significantly lower endogenous thrombin potential and peak thrombin (p<0.001 and p=0.004 respectively) in platelet-rich plasma, a significantly lower endogenous thrombin potential (p=0.004) and peak thrombin (p=0.014) in platelet-poor plasma, and a lower platelet count (p=0.002). On T2 both endogenous thrombin potential and peak thrombin remain significantly lower (p=0.011 and p=0.010) in group 2, measured in platelet-rich plasma but not in platelet-poor plasma. In addition, platelet number remains lower in group 2 after protamine administration (p=0.002).ConclusionsThe key finding is that the Calibrated Automated Thrombography assay, performed preoperatively, provides information predictive for blood loss after cardiac surgery.

Thrombin-generating capacity in patients with von Willebrand’s disease

Background and Objectives on Willebrand’s disease (VWD) is the most common hereditary bleeding disorder. Its severity can be classified on the basis of von Willebrand factor (VWF) and factor VIII (FVIII) plasma levels and according to the clinical relevance of bleeding episodes. However, patients with very low VWF activity may exhibit a mild bleeding tendency. The basis for this heterogeneous clinical expression of the deficit is still poorly understood. We investigated the relationship between thrombin generation and levels of factor VIII, VWF and clinical bleeding tendency. Design and Methods Thrombin generation was measured in platelet-rich (PRP) and platelet-poor plasma (PPP) from 53 patients with VWD. Results We observed a statistically significant higher risk of bleeding in patients with a low thrombin peak in PRP (OR=14.5; 95% CI=5–41.3). Similar results were found in PPP (OR=8.71; 95% CI=3.4–22.3). Two parameters of the thrombin generation curve, peak height and thrombin generation speed (slope), correlated significantly with VWF:RCo and FVIII levels both in PPP and in PRP. Regression analysis showed that thrombin generation was mainly dependent on plasma FVIII activity. Interpretation and Conclusions Our results suggest that the thrombin generation test, in combination with routine FVIII and VWF measurements, could be of interest in the assessment of the individual bleeding risk in patients with VWD.

Inhibition of phospholipid and platelet‐dependent prothrombinase activity in the plasma of patients with lupus anticoagulants

Summary Prothrombinase activity was analysed in the plasma of a series of patients with lupus anticoagulants (LAC). In the presence of purified PS‐PC (20–80%) vesicles the prothrombinase activity triggered by kaolin was retarded by 2–3 min with respect with normal plasma. The maximal values of prothrombinase activity increased by increasing the amount of phospholipid vesicles. However, in the plasma of the patients they were always lower than those of normal plasma at each phospholipid concentration. Platelet‐dependent prothrombinase activity was subsequently investigated. Again, both a delay in appearance and reduced peak values of prothrombinase activity were observed in the plasma of the patients. This inhibition was partially overcome by the addition of an excess of purified phospholipids. Finally, the effect of LAC IgG on platelet rich plasma‐dependent prothrombinase activity was investigated. The main effect observed was a delay of the peak time of prothrombinase activity, while the maximal peaks were affected only by one IgG preparation. We conclude that LAC antibodies can react with both purified negatively‐charged phospholipids and platelet procoagulant phospholipids and inhibit prothrombinase activity in a similar way in both cases.

Measurement of thrombin generation intra-operatively and its association with bleeding tendency after cardiac surgery

INTRODUCTION Patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) are susceptible to haemostatic disturbances. Monitoring the haemostatic capacity by conventional clotting tests is challenging. MATERIALS AND METHODS Thrombin generation (TG) by Calibrated Automated Thrombography, clotting tests and tissue factor pathway inhibitor (TFPI) measurements were performed to describe the relationship between haemostatic changes and alterations in these tests. Blood samples were collected before, during and after CPB. Furthermore, it was investigated whether TG measured intraoperatively, is associated with increased risk of bleeding postoperatively. RESULTS TG diminished significantly (p<0.01) after heparinization in the presence and absence of platelets (37% and 50%) compared to baseline. After the start of CPB, TG elevated and persisted till the end of surgery but remained lower than preoperatively. Activated clotting time increased after heparinization and after the start of bypass compared to baseline (400% and 500%). Anti-FXa activity reduced on the start of CPB compared to the level after heparinization, to almost the baseline value following protamine reversal of heparin. The plasma levels of total and free TFPI elevated 9 and 14 fold during bypass and remained after protamine administration higher than preoperatively. Plasma D-dimer levels reduced (p<0.01) when bypass started. However, a marked elevation was observed in the following time points. TG in platelet-rich plasma measured after heparinization and after the start of CPB associated (p<0.05) with postoperative blood loss. CONCLUSIONS TG can be determined during CPB despite the high heparinization level, it reflects the haemostatic capacity better than clotting-based assays and might better predict bleeding when performed intraoperatively.

Thrombin-dependent Incorporation of von Willebrand Factor into a Fibrin Network

Background: von Willebrand factor recruits platelets into a thrombus via interactions with GPIb. Results: The cleavage of fibrinopeptides A/B is critical for the incorporation of VWF in a fibrin network. VWF incorporates independently from FXIII via its C domains. Conclusion: Fibrin-bound VWF is involved in platelet adhesion at a high shear rate. Significance: The role of VWF in platelet-dependent thrombus formation is explained. Attachment of platelets from the circulation onto a growing thrombus is a process involving multiple platelet receptors, endothelial matrix components, and coagulation factors. It has been indicated previously that during a transglutaminase reaction activated factor XIII (FXIIIa) covalently cross-links von Willebrand factor (VWF) to polymerizing fibrin. Bound VWF further recruits and activates platelets via interactions with the platelet receptor complex glycoprotein Ib (GPIb). In the present study we found proof for binding of VWF to a fibrin monomer layer during the process of fibrinogen-to-fibrin conversion in the presence of thrombin, arvin, or a snake venom from Crotalus atrox. Using a domain deletion mutant we demonstrated the involvement of the C domains of VWF in this binding. Substantial binding of VWF to fibrin monomers persisted in the presence of the FXIIIa inhibitor K9-DON, illustrating that cross-linking via factor XIII is not essential for this phenomenon and suggesting the identification of a second mechanism through which VWF multimers incorporate into a fibrin network. Under high shear conditions, platelets were shown to adhere to fibrin only if VWF had been incorporated. In conclusion, our experiments show that the C domains of VWF and the E domain of fibrin monomers are involved in the incorporation of VWF during the polymerization of fibrin and that this incorporation fosters binding and activation of platelets. Fibrin thus is not an inert end product but partakes in further thrombus growth. Our findings help to elucidate the mechanism of thrombus growth and platelet adhesion under conditions of arterial shear rate.

Asthma is associated with enhanced thrombin formation and impaired fibrinolysis

There is evidence that altered blood coagulation and fibrinolysis are involved in the pathogenesis of asthma. Increased thromboembolic risk has been reported in asthmatics.

Thrombin Generation in Zebrafish Blood.

To better understand hypercoagulability as an underlying cause for thrombosis, the leading cause of death in the Western world, new assays to study ex vivo coagulation are essential. The zebrafish is generally accepted as a good model for human hemostasis and thrombosis, as the hemostatic system proved to be similar to that in man. Their small size however, has been a hurdle for more widespread use in hemostasis related research. In this study we developed a method that enables the measurement of thrombin generation in a single drop of non-anticoagulated zebrafish blood. Pre-treatment of the fish with inhibitors of FXa and thrombin, resulted in a dose dependent diminishing of thrombin generation, demonstrating the validity of the assay. In order to establish the relationship between whole blood thrombin generation and fibrin formation, we visualized the resulting fibrin network by scanning electron microscopy. Taken together, in this study we developed a fast and reliable method to measure thrombin generation in whole blood collected from a single zebrafish. Given the similarities between coagulation pathways of zebrafish and mammals, zebrafish may be an ideal animal model to determine the effect of novel therapeutics on thrombin generation. Additionally, because of the ease with which gene functions can be silenced, zebrafish may serve as a model organism for mechanistical research in thrombosis and hemostasis.

Inhibition of Thrombin-Catalyzed Reactions in Blood Coagulation and Platelet Activation by Heparin Fractions in the Absence of Antithrombin III

The antithrombin-III-independent effect of heparin was studied in the following thrombin-catalyzed reactions: activation of purified plasma factor V and partially purified plasma factor VIII:C, generation of factor Va from the platelets and, in the presence of collagen, of the platelet procoagulant activity. Five heparin fractions and a heparinoid were compared to crude heparin. Crude heparin was a more potent inhibitor of these reactions than the fractions or the heparinoid. The inhibitory action of heparin (fractions) appeared to be the result of the formation of a complex between heparin and thrombin that alters the specificity of thrombin towards high molecular weight substrates. The inhibition of these thrombin-dependent feedback reactions in blood coagulation might be of importance in the mechanisms for the dissociation between the antithrombotic and hemorrhagic properties of low molecular weight heparins.

Impaired fibrinolysis and lower levels of plasma α 2 -macroglobulin are associated with an increased risk of severe asthma exacerbations

Recently we have reported that asthma is associated with enhanced plasma thrombin formation, impaired fibrinolysis and platelet activation. In the present study we investigated whether described prothrombotic blood alterations might predispose to thromboembolic events or asthma exacerbations. In 164 adult asthmatics we assessed clinical events during 3-year follow-up and analyzed their associations with measured at baseline prothrombotic blood parameters. Data were obtained from 157 (95.7%) of the asthma patients. We documented 198 severe asthma exacerbations (64/year), which occurred in 53 subjects (34%). These patients were older (p = 0.004), had worse asthma control (p = 0.02) and lower spirometry values (p = 0.01), at baseline. Interestingly, this subgroup had longer clot lysis time (CLT), as well as lower α2-macroglobulin (p = 0.038 and p = 0.04, respectively, after adjustment for potential confounders). Increased CLT and lower α2-macroglobulin were demonstrated as independent predictors of asthma exacerbation in multiple regression model. Moreover, we documented two episodes of deep vein thrombosis (1.3%), and eight acute coronary syndromes (5.1%). Patients who experienced thromboembolic events (n = 10, 6.4%, 2.1%/year) had lower α2-macroglobulin (p = 0.04), without differences in efficiency of fibrinolysis and thrombin generation. Impaired fibrinolysis and lower levels of α2-macroglobulin might predispose to a higher rate of asthma exacerbations, suggesting new links between disturbed hemostasis and asthma.

Molecular interaction and transport limitation in macromolecular binding to surfaces.

Binding of macromolecules to surfaces, or to surface-attached binding partners, is usually described by the classical Langmuir model, which does not include interaction between incoming and adsorbed molecules or between adsorbed molecules. The present study introduces the “Surfint” model, including such interactions. Instead of the exponential binding behaviour of the Langmuir model, the Surfint model has tanh binding equations, as confirmed by a random sequential adsorption (RSA) computer simulation. For high binding affinity, sorption kinetics become diffusion-limited as described by the existing unstirred-layer model “Unstir”, for which we present the exact analytical solution of its binding equations expressed in Lambert W-functions. Low-affinity binding of thrombin on heparin, and high-affinity binding of prothrombin on phospholipid vesicles, were measured by ellipsometry and were best described by the Surfint and Unstir models, respectively.