Heparins: A Shift of Paradigm.

Heparins inhibit the thrombin forming capacity of plasma, i. e., the endogenous thrombin potential (ETP), by their anti-thrombin (aIIa) activity, the anti-factor Xa (aXa) activity is of minimal importance.

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.

Linear diffusion of thrombin and factor Xa along the heparin molecule explains the effects of extended heparin chain lengths.

Question: How does the size of the heparin moiety in the anti-thrombin (AT)-heparin complex influence its anticoagulant properties?

Approach: Of 52 heparin fractions of precise Mr between 2800 and 37,000 we determined the dissociation constant (Kd) of the binding of the enzyme to the AT-heparin complex and the decay constant (kdec) of thrombin and factor Xa at 1 microM of that complex.

Results: The Kd of thrombin or factor Xa is constant when expressed in terms of the concentration of sugar units, i.e.

Thrombin generation, a function test of the haemostatic-thrombotic system.

By the use of a fluorogenic thrombin substrate and continuous calibration of each individual sample, it is now possible to obtain a thrombin generation (TG) curve (or thrombogram) in plasma, with or without platelets, in an easy routine procedure at high throughput and with an acceptable experimental error (<5%). Evidence is growing that the parameters of the thrombogram, and notably the area under the curve (endogenous thrombin potential, ETP), are useful in assessing bleeding- or thrombotic risk and its modification by antithrombotic- or haemostatic treatment. Available data strongly suggest that conditions (congenital, acquired, drug-induced) that increase TG all cause a thrombotic tendency and that conditions that decrease TG prevent thrombosis but, beyond a limit, cause bleeding.

Low molecular weight activated protein C inhibitors as a potential treatment for hemophilic disorders.

The synthesis and evaluation of inhibitors of activated protein C (aPC) are reported. This serine protease is partly responsible for the degradation of factor VIIIa, involved in the regulation of bleeding in hemophilia A. Benzamidine-containing derivatives were found to be potent aPC inhibitors, some of them showing selectivity against the procoagulant protease thrombin.

Evaluation of thrombin generating capacity in plasma from patients with haemophilia A and B.

In haemophilia patients, a relationship is usually observed between the clinical expression of the disease and plasmatic factor VIII/factor IX (FVIII/FIX) activity. However, it is known from clinical experience, that some haemophilia patients, despite similar FVIII/FIX plasma levels, could exhibit different bleeding phenotype. After determining preanalytical test conditions, we evaluated the thrombin generation capacity from haemophilia plasma samples in various conditions and the potential usefulness of thrombin generation test (TGT) in haemophilia patients.

Thrombin generation assays: accruing clinical relevance.

Purpose Of Review: After decades of near oblivion, thrombin generation is being revived as an overall function test of the plasmatic coagulation system in platelet-poor plasma (PPP). In platelet-rich plasma (PRP) it assesses platelet procoagulant functions as well.

Recent Findings: The recently developed use of special fluorogenic thrombin substrates allows monitoring of thrombin concentration in clotting PPP and PRP on line in up to 24 parallel samples.

Factor XI-dependent reciprocal thrombin generation consolidates blood coagulation when tissue factor is not available.

Objective: Feedback activation of factor XI by thrombin is a likely alternative for tissue factor-dependent propagation of thrombus formation. However, the hypothesis that thrombin can initiate and propagate its formation in a factor XI-dependent and platelet-dependent manner has not been tested in a plasma milieu.

Methods And Results: We investigated thrombin generation in recalcified platelet-rich plasma activated with varying amounts of thrombin or factor VIIa.

[The Choay domain -- the structure responsible for the anticoagulant action of heparins].

We describe the common structural basis for the anticoagulant action of the many different heparins available to the clinician. From different types of heparin we prepared fractions of virtually single molecular weight. We determined the molar concentration of material (HAM) containing the antithrombin (AT) binding pentasaccharide (A-domain), the specific catalytic activity in thrombin- and factor Xa inactivation and the capacity to inhibit thrombin generation (TG).

Calibrated automated thrombin generation in frozen-thawed platelet-rich plasma to detect hypercoagulability.

To enhance the practical applicability of the calibrated automated thrombogram (CAT) we investigated whether frozen-thawed platelet-rich plasma (ft-PRP) can be used to assess the function of the protein C inhibitory pathway, while preserving the natural phospholipid composition. Recalcified ft-PRP triggered with 0.5 pM recombinant human tissue factor shows a median thrombin potential of 1,779 nM x min, against 1,576 nM x min for fresh PRP.

Calibrated automated thrombin generation measurement in clotting plasma.

Calibrated automated thrombography displays the concentration of thrombin in clotting plasma with or without platelets (platelet-rich plasma/platelet-poor plasma, PRP/PPP) in up to 48 samples by monitoring the splitting of a fluorogenic substrate and comparing it to a constant known thrombin activity in a parallel, non-clotting sample. Thus, the non-linearity of the reaction rate with thrombin concentration is compensated for, and adding an excess of substrate can be avoided. Standard conditions were established at which acceptable experimental variation accompanies sensitivity to pathological changes.

Thrombinography shows acquired resistance to activated protein C in patients with lupus anticoagulants.

In patients with lupus anticoagulants (LA), acquired resistance to activated protein C (APC) is difficult to demonstrate with clot-based assays due to the presence of the anticoagulant. Via the conversion of a fluorogenic substrate (thrombinography), we monitored the complete process of thrombin formation and decay and its delimitation by the protein C system in eight consecutive LA-patients without anticoagulant therapy and non-carriers of the V Leiden polymorphism. Thrombin generation was triggered in platelet-poor and platelet-rich plasma by recalcification in the presence of a low concentration of tissue factor.

The thrombogram in rare inherited coagulation disorders: its relation to clinical bleeding.

We investigated the relation between clotting factor concentration, the parameters of the thrombin generation curve (the thrombogram) and the severity of clinically observed bleeding in patients with congenital deficiency of prothrombin (n = 21), factor V (n = 22), factor VII (n = 22), factor X (n = 10), factor XI (n = 7) and factor XII (n = 6). The parameters used were: area under the curve (endogenous thrombin potential, ETP), peak concentration of thrombin attained and lag time before manifest formation. Peak height and ETP varied linearly with the concentration of prothrombin.