Aleksandra Antovic

A Simple and Rapid Laboratory Method for Determination of Haemostasis Potential in Plasma: II. Modifications for Use in Routine Laboratories and Research Work

To offer a suitable method for use in routine laboratories and research work, some modifications were made in the assay of overall haemostatsis potential (OHP) we developed earlier. Thrombin in a decreased dose with or without tissue-type plasminogen activator was added to plasma for initiation of fibrinogen clotting. Areas under two fibrin-aggregation curves i.e., above-mentioned OHP and overall coagulation potential (OCP) were thus created. A difference between the two parameters reflects the overall fibrinolysis potential (OFP), calculated by ((OCP-OHP)/OCP) x 100%. To obtain reference ranges, investigations were performed in 142 healthy adults of different ages and in 29 healthy women with a normal pregnancy. In 16 patients suffering from coronary heart disease (CHD), OCPs and OHPs increased but OFPs decreased. In 10 pre-eclamptic women with moderate enhancement of OCP, the OHPs became noticeably higher in most while the OFPs lower. Extremely low or undetectable levels of OHP and OCP were shown in samples of Factors VIII-, IX-, VII-, V-, X- or II-deficient plasma. In 13 healthy volunteers treated with acetylsalicylic acid (ASA), OHPs expectedly declined during the administration and rose again after withdrawal. The above findings demonstrate that the modifications in the present study have rendered the method more effective for detecting haemostatic changes and relevant for monitoring treatments.

Atorvastatin reduces thrombin generation and expression of tissue factor, P-selectin and GPIIIa on platelet-derived microparticles in patients with peripheral arterial occlusive disease

We investigated the effects of statin treatment on platelet-derived microparticles (PMPs) and thrombin generation in atherothrombotic disease. Nineteen patients with peripheral arterial occlusive disease were randomised to eight weeks of treatment with atorvastatin or placebo in a cross-over fashion. Expression of GPIIIa (CD61), P-selectin (CD62P), tissue factor (TF, CD142) and phosphatidylserine (PS; annexin-V or lactadherin binding) was assessed on PMPs. Thrombin generation in vivo was assessed by measurement of prothrombin fragment 1+2 in plasma (F1+2) and ex vivo by using the calibrated automated thrombogram (CAT). During atorvastatin treatment, expression of TF, P-selectin and GPIIIa was significantly reduced vs. placebo (p<0.001 for all). No effect on annexin-V or lactadherin binding was seen. Thrombin generation was significantly reduced during atorvastatin as assessed by both the CAT assay (p<0.001) and by measurements of F1+2 (p<0.01). Subsequent in vitro experiments showed that when TF on microparticles (MPs) was blocked by antibodies, the initiation of thrombin generation was slightly but significantly delayed. Blocking PS on MPs using annexin-V or lactadherin resulted in almost complete inhibition of thrombin generation. In conclusion, atorvastatin reduces thrombin generation and expression of TF, GPIIIa and P-selectin on PMPs in patients with peripheral vascular disease. Microparticle-bound TF slightly enhances initiation of thrombin generation whereas negatively charged surfaces provided by MPs or lipoproteins could reinforce thrombin generation. Statins may inhibit initiation of thrombin generation partly through a microparticle dependent mechanism but the main effect is probably through reduction of lipoprotein levels.

Atorvastatin has antithrombotic effects in patients with type 1 diabetes and dyslipidemia.

INTRODUCTION Diabetes is a prothrombotic state involving a more thrombogenic fibrin network. In the present study we investigated the effects of lipid-lowering therapy with atorvastatin on fibrin network structure and platelet-derived microparticles in patients with type 1 diabetes and dyslipidemia. MATERIALS AND METHODS Twenty patients were treated with atorvastatin (80 mg daily) or placebo during 2 months in a randomized, double-blind, cross-over study. Fibrin network permeability, expression of glycoprotein IIIa, P-selectin and tissue factor on platelet-derived microparticles, plasma endogenous thrombin potential, plasminogen activator inhibitor-1 and tissue plasminogen activator antigen levels were assessed. Additionally, levels of plasma fibrinogen, high-sensitivity C-reactive protein and glycated haemoglobin were measured. RESULTS During treatment with atorvastatin, fibrin network permeability increased (p=0.01), while endogenous thrombin potential and expression of glycoprotein IIIa, P-selectin and tissue factor decreased (p<0.01). In vitro experiments indicated that platelet-derived microparticles influence the fibrin network formation as fibrin network permeability decreased significantly when platelet-derived microparticles were added to normal plasma. Baseline levels of plasminogen activator inhibitor-1 and tissue plasminogen activator antigen as well as plasma fibrinogen and high-sensitivity C-reactive protein were within reference values and not significantly changed during atorvastatin treatment, while glycated haemoglobin increased 0.3% (p<0.001). CONCLUSIONS Novel treatment effects were found in patients with type 1 diabetes and dyslipidemia during atorvastatin therapy, i.e. a more porous fibrin network, to which reduced expression of glycoprotein IIIa, P-selectin and tissue factor on platelet-derived microparticles may contribute. The observed impairment of glycemic control during long-term statin treatment deserves attention.

Modifications of flow measurement to determine fibrin gel permeability and the preliminary use in research and clinical materials

Our earlier investigations employing a flow measurement have yielded intriguing findings as to what governs the fibrin network porosity. To make the method suitable for use by more groups with various laboratory conditions, sample materials or study purposes, we simplified the essential equipment and thereby minimized the sample volume to 250 μl in comparison with the need for 3000 μl in the previous method. To assess whether the fibrin gel permeability depends on changes in thrombin generation potential and/or fibrinogen clotting property, different concentrations of thrombin with or without frozen–thawed platelets, serving as phospholipids, were used. The platelets and 0.05 IU/ml thrombin were added to plasma samples from patients with previous myocardial infarction. The fibrin gel permeability, expressed as Darcy constant (Ks), was decreased compared with that in controls, supporting findings about high risk of thromboembolism in this disease due to increases of thrombin activity and fibrinogen function. When 0.4 IU/ml thrombin was used in samples provided by 10 healthy individuals treated with acetysalicylic acid, Ks levels were increased during versus before therapy. Since almost no thrombin generation was found in the samples with the higher dose of exogenous thrombin, we considered that modifications in fibrinogen clotting property by acetysalicylic acid rendered the fibrin network more permeable. In summary, as the reproducibility remains satisfactory (coefficient of variation < 10%) despite aforementioned modifications in the equipment and reagents, any interested laboratory ought to be able to repeat the method. Assays of fibrin permeability in such a simple way may help to determine the fibrin clot stability in pathological/pharmacological studies, and probably serve as a tool to estimate thromboembolism risk in clinical materials, such as patients with cardiovascular diseases.

The Overall Hemostasis Potential: A Laboratory Tool for the Investigation of Global Hemostasis

A simple and rapid global haemostatic assay for determination of the overall hemostasis potential (OHP) in plasma represents a new approach in detecting alterations in the delicate balance between coagulation and fibrinolysis. The assay is based on repeated spectrophotometric registration of the fibrin-aggregation curve in platelet-poor plasma containing small amounts of exogenous thrombin, tissue-type plasminogen activator, and calcium. The overall coagulation potential and overall fibrinolytic potential are supplementary parameters of OHP, providing details of underlying changes in coagulation and/or fibrinolysis. The OHP assay was evaluated in connection with hypercoagulation in normal pregnancy, preeclampsia, some thrombophilias, coronary heart disease, diabetes, stroke, and vascular surgery as well as with hypocoagulability, especially in patients with hemophilia A or B. Preliminary results also indicate the possible usefulness of the assay in monitoring anticoagulant treatments. Large prospective clinical trials are needed before the method can be recommended for routine clinical application.

Studies of microparticles in patients with the antiphospholipid syndrome (APS)

Objectives: To study circulating platelet, monocyte and endothelial microparticles (PMPs, MMPs and EMPs) in patients with antiphospholipid syndrome (APS) in comparison with healthy controls. Material and method: Fifty-two patients with APS and 52 healthy controls were investigated. MPs were measured on a flow cytometer (Beckman Gallios) and defined as particles sized < 1.0 µm, negative to phalloidin, positive to lactadherin and positive to either CD42a (PMPs), CD144 (EMPs) or CD14 (MMPs). Exposure of CD142 (TF) was measured on CD144 positive MPs. Results: Total number of MPs (i.e. lactadherin positive particles) was higher in APS patients versus controls (p < 0.001). An increased number of EMPs (p < 0.001), increased TF-positive EMPs (p < 0.001) and increased MMPs (p < 0.001) were also observed. PMP numbers did not differ between the groups. None of the MP types differed in numbers between obstetric and thrombotic APS patients. Conclusion: We observed a high number of EMPs expressing TF in APS patients. The numbers of MMPs and total EMPs were also higher as compared with healthy controls but in contrast to previous reports, the number of PMPs did not differ between groups.

Increased hemostasis potential persists in women with previous thromboembolism with or without APC resistance

Summary.  Background: Activated thrombin generation and depressed fibrinolysis due to the presence of activated protein C (APC) resistance with or without factor (F)V Leiden mutation are associated with development of deep venous thrombosis (DVT). Objective: A better understanding of the mechanism behind the risk of recurrence of DVT, using our new, recently developed assay of overall hemostasis potential (OHP). Patients and methods: Levels of OHP, as well as APC resistance and FV Leiden mutation, were determined in 88 women (cases) who had previously experienced DVT in connection with pregnancy, and in 25 young healthy individuals (controls). Clotting time and clot lysis time were also investigated. Results: OHP levels in the patients were increased compared with the controls. In the cases with APC resistance and the Leiden mutation this imbalance in hemostasis potential was more severe than in those without. The group with the more severe imbalance had shorter clotting times and longer clot lysis times. Conclusions: A procoagulant state perseveres in patients with a history of pregnancy‐related DVT, even after the symptomatic phase is over. The mechanisms behind such an imbalance in overall hemostasis are enhanced thrombin generation and depressed fibrinolysis. These findings may underscore the need for thromboprophylaxis to prevent recurrence of thromboembolism in risk situations.

Does recombinant factor VIIa, apart from overall hemostasis, regulate TAFI dependent fibrinolysis? In vitro analysis using overall hemostasis potential (OHP) assay

Using the recently developed overall hemostatic potential (OHP) assay, we investigated the effects of different concentrations of recombinant factor VIIa on overall hemostasis and on thrombin activatable fibrinolysis inhibitor (TAFI) dependent fibrinolysis in different factor deficient plasmas. rFVIIa increased OHP in FV, FVIII and FIX deficient plasmas but not up to the levels in normal pooled plasma (NPP). Maximal levels were found at 2.4 microg/mL of rFVIIa, while higher doses did not further increase OHP. In FXII deficient plasma, OHP is higher than in NPP and addition of rVIIa further increased it. Even very high concentrations of rFVIIa (9.6 microg/mL) did not induce a significant increase of OHP in NPP. Higher concentrations of rVIIa down-regulated fibrinolysis in FVIII, FIX and FXI deficient plasmas to values obtained in NPP. Using potato tuber carboxy-peptidase inhibitor (PTCI), a specific inhibitor of TAFI, it was found that TAFIs influence on fibrinolysis down-regulation in FVIII and FIX deficient plasmas increased after addition of higher concentrations of rFVIIa. From this in vitro study it seems that rFVIIa in a concentration of 2.4 microg/mL improves overall hemostasis in FV, FVIII and FIX deficient plasmas. rFVIIa, even at very high (supra-therapeutic) concentrations, does not induce hypercoagulability either in NPP or in deficient plasmas. Higher concentrations of rVIIa induce, at least partly, TAFI dependent down-regulation of fibrinolysis in FVIII and FIX deficient plasmas. These results, together with some previous ex vivo studies, point to OHP assay as a possible diagnostic tool for evaluating the hemostatic effects of rFVIIa.

Identifying hypocoagulable states with a modified global assay of overall haemostasis potential in plasma.

To test the sensitivity of the global assay of overall haemostasis potential (OHP) in detecting hypocoagulation, the OHP was assayed in plasma containing exogenous thrombin (0.04 IU/ml), tissue-plasminogen activator (330 ng/ml), Ca2+ and a platelet reagent. Commercial plasmas with factor II, V, VIII, IX, X, XI, XII or VII deficiency were mixed with normal plasma in different proportions to imitate different severities. Samples from patients with haemophilia and factor XII deficiency were also examined. No clot was found in the absence of factor II/factor X, indicating that the tiny dose of thrombin worked solely as a trigger for the intrinsic pathway activation. Changed levels of the investigated coagulants, apart from factor XII, influenced the outcome. OHPs were decreased in patients with haemophilia but were unchanged or even increased in those with factor XII deficiency. This modified OHP method may therefore be useful for estimating the bleeding tendency in haemophilic patients and to find suitable doses and intervals for prophylactic treatment. It may also be of use in investigations of the effect of antifibrinolytic drugs as well as for identifying a thrombotic tendency in patients with factor XII deficiency. For detection of other coagulation factor deficiencies, our investigations with the commercial plasmas suggest that the OHP assay is also valuable, especially when the intrinsic pathway of the coagulation cascade is impaired.

Overall haemostatic potential can be used for estimation of thrombin-activatable fibrinolysis inhibitor-dependent fibrinolysis in vivo and for possible follow-up of recombinant factor VIIa treatment in patients with inhibitors to factor VIII

Summary. Thrombin generation induced by recombinant factor VIIa (rFVIIa) in patients with haemophilia and/or inhibitors to factor VIII/IX could enhance generation of thrombin‐activatable fibrinolysis inhibitor (TAFI), a recently described link between coagulation and fibrinolysis. TAFI is unstable and it is not easy to measure its active form in vivo. Overall haemostatic potential (OHP) is a novel method for haemostasis estimation, based on determination of the fibrin aggregation curve in which tiny amounts of thrombin are used for activation of clotting. We measured OHP in six patients with inhibitors to factor VIII before injection of rFVIIa and 10 and 120 min thereafter. Overall fibrinolytic potential (OFP) and clot lysis time (CLT) analysed by this method could be used for indirect estimation of TAFI generation. We found no change in pro‐TAFI and total TAFI antigen before and after treatment with rFVIIa. OHP was almost undetectable before treatment but increased into the range of normal pooled plasma 10 and 120 min after rFVIIa treatment, as did CLT. However, after addition of potato tuber carboxypeptidase inhibitor, a specific inhibitor of TAFI, the shortening of CLT was lower than that in NPP. OFP was increased in patient plasma both 10 and 120 min after treatment compared with NPP. There was a strong positive correlation between pro‐TAFI concentration and shortening of CLT after PTCI addition and a negative correlation between pro‐TAFI concentration and OFP 10 min after rFVIIa injection. Thus, rFVIIa normalizes OHP and CLT 10 min after injection. While this improvement slightly decreases, but still exists after 2 hours, it suggests efficacy in bleeding prevention using a protocol based on rFVIIa administration every 2 hours.