Mario Koksch

Paradoxical hyperfibrinolysis is associated with a more intensely haemorrhagic phenotype in severe congenital haemophilia

Summary. To elucidate potential causes for differing bleeding phenotypes of haemophilic patients of identical degree of coagulation factor deficiency, we investigated 21 male patients with severe haemophilia. Median annual coagulation factor demand and the extent of haemophilic arthropathy were used to discriminate between intensely and less intensely haemorrhagic phenotypes. Haemophiliacs with a median annual coagulation factor demand of 800 IU per kg bodyweight or more and with three or more joints affected by haemophilic arthropathy represented the intensely haemorrhagic phenotype group; all other patients comprised the less intense group. The discriminator values represent the respective medians of the overall group. The results of activated partial thromboplastin time, endogenous thrombin potential, pro‐ and anticoagulant factor analysis did not differ between the two groups. Median tissue‐type plasminogen activator concentration (TPA) was elevated significantly in haemophiliacs with an intensely haemorrhagic phenotype, as was the activity of the thrombin‐activatable fibrinolysis inhibitor. Median activity of the plasminogen activator inhibitor 1 (PAI 1) and the concentration of TPA–PAI 1 complexes were increased to approximately double those in nonsevere haemophiliacs. Coexistent congenital thrombophilia was found significantly more often in the less intensely haemorrhagic group. Thus, increased stimulation of the fibrinolytic system was associated with a more intensely haemorrhagic phenotype in our patients. We hypothesize that ineffective haemophilic haemostasis in response to trauma evokes a protracted stimulation of the entire haemostatic system, including costimulation of fibrinolysis. The absence of coexistent congenital thrombophilia predisposes to excess stimulation of fibrinolysis, which cannot be downregulated effectively due to the dysfunctional intrinsic pathway. The association of a more intensely haemorrhagic phenotype with a paradoxical hyperstimulation of the fibrinolytic system resembles a vicious circle, where bleeding seems to cause predisposition to more bleeding.

The platelet function defect of paroxysmal nocturnal haemoglobinuria

Paroxysmal nocturnal haemoglobinuria (PNH) is a rare, acquired stem cell disorder, characterised by an abnormal susceptibility of red blood cells to complement induced lysis, resulting in repeated episodes of intravascular haemolysis and haemoglobinuria, thromboembolic events at atypical locations and, to a much lesser extent, bleeding complications. Platelet function is assumed to be abnormal, however, a defect has not yet been characterised and underlying mechanisms remain elusive. To explore these issues, we investigated platelet function in PNH patients using assays for clot formation under low and high shear force (thrombelastography and PFA100® device), adhesion to glass beads in native whole blood (Hellem method), aggregometry using various agonists (Born method), and flow cytometric assays for baseline and agonist-induced surface expression density of α-granule (CD62P) and lysosomal granule proteins (CD63), ligand binding to surface receptors (thrombospondin), and expression density of activation-induced neoepitopes of the fibrinogen receptor complex (PAC-1). Platelet PNH clone size determined by CD55 and CD59 labelling was compared to the clone sizes of granulocytes, monocytes, erythrocytes, and reticulocytes. A profound reduction of platelet reactivity was observed in PNH patients for all ‘global function’ assays (clot formation, adhesion, aggregation). Platelet hyporeactivity was confirmed using flow cytometric assays. Whereas baseline levels of flow cytometrically determined platelet activation markers did not differ significantly between controls and PNH patients, agonist-induced values of all markers were distinctly reduced in the PNH group. Moreover, significantly reduced white blood cell counts (3.1/nl vs. 5.9/nl), haemoglobin values (9.5 vs. 14.3/g per dl), and platelet counts (136 vs. 219/nl) delineate profound tricytopenia in PNH patients. The fraction of particular cell types lacking the surface expression of GPI-anchored glycoproteins is referred to as the respective PNH clone; median PNH clone sizes of cells with short life spans (reticulocytes, platelets, granulocytes) was 50–80% of total cell populations compared to 20% of red blood cells. The results of our laboratory investigations show, that in PNH, reduced platelet counts coincide with reduced platelet reactivity. The foremost clinical complication in PNH, however, is venous thromboembolism, very probably induced by an activated and dysregulated plasmatic coagulation system. From these seemingly contradictory findings we infer, that part of the platelet hyporeactivity is probably due to reactive downregulation of platelet function in response to chronic hyperstimulation. The overall result is thought to be an unsteady balance, associated with thromboembolism in a larger proportion of patients, and with bleeding in a smaller proportion.

Synthesis of cyclic RGD-peptides containing β-amino acids

The solid phase synthesis of cyclic RGD-peptides containing β-amino acids according to two different protocols is described. The second strategy allows multiple or combinatorial syntheses of this type of cyclic peptides, because it enables backbone cyclization while the RGD-peptide is still bound to the resin. The newly synthesized RGD-peptides were characterized by MALDI-TOF MS and NMR and their physiological activity was determined by aggregometry.

Fibrinogen receptor antagonists induce conformational changes of the human platelet glycoprotein IIb

Controversial results have been reported concerning the ability of fibrinogen receptor antagonists (fibans) to induce conformational changes in the fibrinogen receptor after binding to it as the initial step of fibrinogen binding and platelet activation.

Flow Cytometric Analysis of Platelet Function in Micro- and Macroangiopathy.

Aim of this study was the development of a flow cyiometric assay to detect platelet activation using in vitro stimulation with physiological agonists. Therefore, in healthy subjects and insulin-dependent diabetics with and without microangiopathy (retinopathy and/or nephropathy) the surface expression density of fibrinogen receptor complex gpIIb/IIIa (CD41), von Willebrand factor complex gpIb/IX (CD42b), ctgranule protein GMP-140 (CD62P), and of lysosomal protein gp53 (CD63) were measured ex vivo and after in vitro stimulation using ADP and thrombin receptor activator peptide 6 (TRAP-6). Best discrimination (p<0.05) between diabetics and controls were observed in the surface expression density of GMP-140 after maximum stimulation with the weak agonist ADP (20 μπιοΐ/ΐ) and sub-maximal stimulation with the strong agonist TRAP-6 (5 μπιοΐ/ΐ). Induced platelet aggregometry using collagen, ristocetin, ADP, and TRAP-6 and plasma concentrations of platelet factor 4 and -thromboglobulin failed to show any differences between the groups. GMP-140 levels were analyzed in a dual-color whole blood assay (CD41-PE and CD62P-F1TC) in 50 healthy controls and 60 patients suffering from peripheral arterial occlusive disease. Highly significant differences (p<0.005) were found between the two groups in the ex vivo as well as in the in vitro stimulated samples (mean fluorescence ±SEM ex vivo: 0.43 ±0.02 vs. 0.50 ±0.02; 5 μπιοΐ/ΐ ADP 0.93 ±0.03 vs. 1.30 ±0.04; 10 μιηοΐ/ΐ TRAP-6 3.30 ±0.17 vs. 3.91 ±0.16; all p<0.005). In conclusion, the in vitro platelet stimulation assay is an easy, fast, and sensitive tool for the detection of platelet hyperreactivity.