Beate Eichelberger

Loss of high-molecular-weight von Willebrand factor multimers mainly affects platelet aggregation in patients with aortic stenosis

Severe aortic stenosis is associated with a haemostatic abnormality that resembles acquired von Willebrand syndrome type 2. It is assumed that high shear conditions render large von Willebrand factor (VWF) multimers accessible to cleavage by ADAMTS-13. However, whether loss of these large multimers affects platelet function by impairing adhesion, aggregate formation, or both has not been evaluated in clinical studies. We prospectively enrolled 47 patients with severe aortic stenosis, and studied them prior to aortic valve surgery and at a median of six months after valve replacement. We investigated levels of large VWF multimers, platelet function under high shear conditions, and residual response to suboptimal concentrations of ADP to express P-selectin. As expected, there was a significant reduction of VWF large multimers before surgery that resolved thereafter in most patients (p<0.0001). The closure time of the ADP cartridge of the PFA-100 was also corrected in most patients after the operation (p<0.0001). We used the cone and plate(let) analyser Impact-R to differentiate between adhesion and aggregation. Both adhesion (p=0.03) and ADP-inducible platelet aggregation (p=0.002) improved considerably after valve replacement. Consequently, ADP-inducible expression of P-selectin was higher after valve replacement (p=0.001). We conclude that reduced levels of large VWF multimers associated with aortic stenosis lead to impairment of both adhesion and, especially, ADP-inducible platelet aggregation.

Monitoring survival and function of transfused platelets in Bernard‐Soulier syndrome by flow cytometry and a cone and plate(let) analyzer (Impact‐R)

BACKGROUND: Bernard‐Soulier syndrome (BSS) patients may repeatedly require transfusion of platelets (PLTs). The hemostatic competence of transfused PLTs requires monitoring.

In vitro platelet function of platelet concentrates prepared using three different apheresis devices determined by impedance and optical aggregometry

BACKGROUND: Testing the functional capacity of platelets (PLTs) from platelet concentrates (PC) is a main issue in transfusion medicine. Therefore, the aim was to study agonist‐inducible PLT aggregation of PLTs obtained by three apheresis devices. A semiautomated impedance aggregometry‐based whole‐blood method (multiplate electrode PLT aggregometry [MEA]) was modified for the use of PLTs from PC and data were compared with light transmission aggregometry (LTA).

Impact of variables of the P-selectin – P-selectin glycoprotein ligand-1 axis on leukocyte-platelet interactions in cardiovascular disease

The formation of leukocyte-platelet aggregates (LPA), through the P-selectin - P-selectin glycoprotein ligand (PSGL)-1 axis, plays a pivotal role in atherothrombosis. In order to investigate the influence of platelet (pP-selectin) and soluble P-selectin (sP-selectin), and of variations in the genes encoding for P-selectin (SELP) and PSGL-1 (SELPLG) on LPA formation, we assessed monocyte (MPA)- and neutrophil-platelet aggregates (NPA) as well as pP-selectin by flow cytometry in 263 patients undergoing angioplasty and stenting. sP-selectin was determined by ELISA, the SELP Pro715 allele and the SELPLG Ile62 allele were determined by allele specific PCR. The Pro715 allele was significantly associated with lower levels of in vivo pP-selectin and sP-selectin, while agonists´ inducible pP-selectin was not influenced by the Pro715 allele. PP-selectin was significantly associated with MPA and NPA formation. The in vivo formation of MPA and NPA depended to 19 % and 7.4 %, respectively, on in vivo pP-selectin, irrespective of the Pro715 allele and the Ile62 allele carrier status. TRAP-6 inducible MPA and NPA depended to 34 % and 27 %, respectively, on TRAP-6 inducible pP-selectin, but were independent of the Pro715 allele carrier status. Carriers of the Ile62 allele showed a stronger correlation between TRAP-6 inducible pP-selectin and TRAP-6 inducible MPA/NPA than non-carriers. Furthermore, TRAP-6 inducible NPA were higher in Ile62 allele carriers, which suggests higher thrombin sensitivity. In conclusion, our findings point to the significant role of pP-selectin for MPA and NPA formation, while other variables like sP-selectin, the SELP Pro715 allele and the SELPLG Ile62 allele have less influence.

IgG titer, subclass, and light-chain phenotype of pregnancy-induced HPA-5b antibodies that cause or do not cause neonatal alloimmune thrombocytopenia.

BACKGROUND: The most common pregnancy‐induced platelet‐specific antibody is HPA‐5b. Neonatal alloimmune thrombocytopenia that results from anti‐HPA‐5b may cause severe hemorrhage in only a few infants, but the sequelae for the affected children can be severe. It is therefore essential that infants at risk for neonatal alloimmune thrombocytopenia are identified.

Sex differences of leukocyte–platelet interactions and on-treatment platelet reactivity in patients with atherosclerosis

OBJECTIVE To investigate differences of platelet activation and on-treatment residual platelet reactivity between female and male patients with atherosclerotic cardiovascular disease. METHODS We compared P-selectin expression, activated glycoprotein (GP) IIb/IIIa and leukocyte-platelet aggregates (LPA) by flow cytometry between 110 female and 206 male patients undergoing angioplasty and stenting. On-treatment residual platelet reactivity was determined by two test systems. RESULTS The expression of P-selectin and GPIIb/IIIa did not differ significantly between female and male patients. In contrast, females showed a significantly more pronounced formation of LPA in vivo, in response to thrombin receptor-activating peptide-6 and in response to adenosine diphosphate. Further, high LPA were seen more frequently in female patients. Finally, protease-activated receptor (PAR)-1 mediated platelet reactivity by both assays was significantly higher in females. CONCLUSION Female sex is associated with a more pronounced formation of LPA and increased PAR-1 mediated platelet reactivity in atherosclerotic cardiovascular disease.

Cross validation of aspirin effect in healthy individuals by Impact-R and PFA-100: A double blind randomized placebo controlled trial

The main objective of this study was to compare testing for aspirin response in healthy volunteers by two high shear methods in a randomized double blind placebo controlled study. Seventeen healthy male individuals were randomized for aspirin 160 mg per day for 7–10 days, and 20 age matched controls for placebo for the same period. At study entry and 7–10 days thereafter we determined high shear-induced platelet adhesion to polystyrene after pre-incubation with arachidonic acid using the Cone and Plate(let) analyzer (Impact-R), and the closure time of collagen/epinephrine cartridges obtained by the PFA-100 (CEPI-CT). Platelet adhesion to polystyrene after preincubation with arachidonic acid was median 3.7% (range 0.6–8.0) before study entry and median 6.7% (range 2.8–11.0) after 7–10 days of aspirin (p < 0.001). Changes were not significant in the placebo group. By the PFA-100 CEPI-CT was median 211 s (range 130–300 s) before aspirin, and 300 s in all individuals taking aspirin for 7–10 days (p < 0.001). Post-treatment data obtained by the Impact-R and PFA-100 were discordant in seven cases from the placebo group, and in one subject on aspirin. The response to aspirin varied considerably among healthy individuals, but both methods were suitable to demonstrate the aspirin effect. There was, however, a significant level of absent concordance between the tests. Since the trial design cannot provide data on the specificity of the different tests, only clinical experience can determine their usefulness.

Inhibitory Activity of Aspirin on von Willebrand Factor-Induced Platelet Aggregation

The effect of aspirin (ASA) on vWF induced platelet - platelet interaction is unknown. We therefore tested the response of platelets to von Willebrand factor (vWF) coated beads induced platelet aggregation before and after i.v. and oral ASA. 1000 mg ASA was infused to 10 healthy individuals and after a wash-out period 7 volunteers received 100 mg ASA orally over a period of 11 days. Prior to ASA and in regular intervals thereafter we tested the reactivity to vWF-coated beads to assess platelet adhesion/aggregation and the fade-out time of ASA effects on platelets. Considerable interindividual variability in response to vWF-coated beads was observed, both before ASA and after treatment with ASA. The maximal response to vWF-coated beads (Tmax), the time lag, and the slope of the curve were significantly affected by i.v. ASA, whereas 100 mg of ASA had only inconstant effect on Tmax and slope. The absolute reduction of Tmax after ASA depended on the pre-ASA level, while the percentage of the reduction was similar in all individuals. Thus, platelet aggregation induced by vWF-coated beads is impaired by ASA. Furthermore, our data indicate a large interindividual variability of the response to ASA shortly after treatment induction, which becomes more constant after prolonged treatment.

Platelet-specific markers are associated with monocyte-platelet aggregate formation and thrombin generation potential in advanced atherosclerosis

Platelet activation and thrombin generation are crucial steps in primary and secondary haemostasis. However, both also play major roles in intravascular thrombus formation and therefore in the development of adverse cardiovascular events. In the current study, we first sought to investigate the associations of the platelet biomarkers platelet factor (PF)-4, thrombospondin (TSP)-1, soluble CD40 ligand (sCD40L), and soluble P-selectin (sP-selectin) with each other and with monocyte-platelet aggregate (MPA) formation in 316 patients undergoing angioplasty and stenting. To better understand the interplay between platelet activation and thrombin generation, we subsequently investigated the associations of the platelet biomarkers with thrombin generation potential. The mostly platelet-specific markers PF-4, TSP-1 and sCD40L correlated strongly with each other (all p < 0.001), and the best correlation was observed between PF-4 and TSP-1 (r=0.91). In contrast, sP-selectin, which derives from platelets and endothelial cells, correlated rather poorly with TSP-1 (r=0.12, p=0.04), and did not correlate with PF-4 and sCD40L. While PF-4, TSP-1 and sP-selectin correlated significantly with in vivo MPA formation (all p< 0.001), no such association was found between sCD40L and MPA formation. PF-4, TSP-1 and sCD40L correlated strongly with peak thrombin generation (all p< 0.001) with the best correlation between PF-4 and peak thrombin generation (r=0.55), whereas sP-selectin did not correlate with peak thrombin generation. Likewise, PF-4, TSP-1 and sCD40L correlated significantly with the area under the thrombin generation curve (AUC; all p< 0.01), whereas sP-selectin did not correlate with the AUC. In conclusion, platelet-specific markers are associated with MPA formation and thrombin generation potential in patients with advanced atherosclerosis.

Decreased platelet reactivity in patients with cancer is associated with high risk of venous thromboembolism and poor prognosis

Platelets are suggested to play a crucial role in cancer progression and the prothrombotic state of cancer patients. Here, we aimed to examine the activation status of platelets in cancer patients and investigate their association with risk of death and occurrence of venous thromboembolism (VTE) in a prospective observational cohort study. We measured platelet surface P-selectin, activated glycoprotein (GP) IIb/IIIa and monocyte-platelet aggregate (MPA) formation in vivo and platelet response to ex vivo stimulation with agonists of protease-activated receptor (PAR) -1, -4, and GPVI, by whole blood flow cytometry, before beginning of chemotherapy and repeatedly during the first six months thereafter (total number of samples analysed: 230). Endpoints of the study were occurrence of death or VTE during a two-year follow-up, respectively. Of 62 patients (median age [interquartile range, IQR]: 63 [54-70] years, 48 % female), 32 (51.6 %) died and nine (14.5 %) developed VTE. Association with a higher risk of death was found for lower platelet surface expression of P-selectin and activated GPIIb/IIIa in vivo and in response to PAR-1, -4 and GPVI activation, but not for MPA formation. Furthermore, reduced platelet responsiveness to PAR-1 and GPVI agonists was associated with higher risk of VTE (hazard ratio per decile increase of percentage P-selectin positive platelets: 0.73 [0.56-0.92, p=0.007] and 0.77 [0.59-0.98, p=0.034], respectively). In conclusion, cancer patients with a poor prognosis showed decreased platelet reactivity, presumably as a consequence of continuous activation. Our data suggest that decreased platelet reactivity is associated with increased mortality and VTE in cancer.