Norbert Bender

Differential effects of clivarin and heparin in patients undergoing hip and knee surgery for the generation of anti-heparin-platelet factor 4 antibodies

The pathophysiology of heparin-induced thrombocytopenia (HIT) syndrome is mediated via a heterogeneous group of heparin(s)-platelet factor 4 (H-PF4) complexes bound to their antibodies. These anti-H-PF4 (AHPF4) antibodies that are capable of binding to the FcgammaRIIA receptor [cluster of differentiation (CD) 32] on platelets, resulting in platelet activation, widely vary in their specific activities as platelet activation (functionality). Predisposing factors related to specific pathologic conditions may also contribute to the generation of these antibodies and their relative functionality during HIT syndrome. To understand this phenomenon, a sub-study was carried out in patients undergoing elective total hip and knee replacement surgery (ECHOS Study) and who were treated with unfractionated heparin (UFH) and a low-molecular-weight heparin (LMWH; Clivarin). Approximately 600 patients per arm [UFH=7,500 anti-Xa U twice a day (b.i.d.) subcutaneous (s.c.) and clivarin=4200 U once daily (o.d.) s.c.], age >40 years, received prophylactic treatment for a minimum of 11-14 days. Plasma samples were collected at pre-dose, days 2-4, days 11-14 and at follow-up 6-8 weeks after discharge and were analyzed for AHPF4 antibody titers. Functionality of the enzyme-linked immunosorbant assay (ELISA)-positive AHPF4 antibodies to cause platelet activation was tested by 14C-serotonin release assay (SRA). Both UFH and clivarin treatments in orthopedic surgical patients resulted in a progressive generation of AHPF4 antibodies. The relative prevalence/functionality of AHPF4 antibodies in clivarin arm was markedly lower (two- to threefold, p<0.001) as compared to UFH at each time point. Most of the samples in clivarin group were found to be SRA negative, suggesting the presence of AHPF4 antibodies that did not activate platelets (nonfunctional). Within the UFH arm, the relative prevalence/functionality of AHPF4 antibodies was much higher (p<0.002) in knee group compared to the corresponding hip group. This study, for the first time, reports on the elevated levels of AHPF4 antibodies generated by heparin associated with the pathogenesis of knee surgery. Clinical significance of the differential generation of HIT-associated antibodies remains unexplored at this time.

Stimulation of blood platelets by extracts of subcutaneous tissue.

Abstract If blood is fixed at blood sampling by feeding it directly into glutaraldehyde 15 – 25 % of the platelets show pseudopodes and are sphered, while the majority retains their disc like shape. When the canula was accidentally inserted into the perivascular tissue and then withdrawn to collect venous blood almost all platelets were sphered and showed pseudopodes. Similar morphologic changes could be regularily induced within 1 – 2 seconds by admixing subcutaneous tissue extracts to freshly drawn blood before fixation. The activity which causes these rapid morphologic changes seems to be independent of ADP or collagen and more active. It does not induce aggregation but may be important for the initiation of primary hemostasis. Similar effects were obtained with lysolecithin and monophosphoinositit/phosphatidylcholine.

A hemostasis activating factor (HAF) in subcutaneous tissue extracts: Effects on morphologic platelet changes, platelet retention and platelet aggregation

Abstract Extracts from both human and porcine subcutaneous tissue were obtained by extraction with propanol. After centrifugation the intermediate zone contained potent platelet stimulating activity. Platelet morphology changed by sphering and pseudopode formation within seconds after incubation of these lipoprotein containing tissue extracts with freshly drawn blood. The tissue extracts were active in dilutions up to 1:1000. At room temperature and at 6°C the extracts lost their activity after 14 - 21 days. At −17°C, −60°C or freeze dried they retained their activity for more than 45 days. At 37°C the stimulating effects on platelets in citrated blood was reversible within 10 minutes. The tissue extracts strongly enhanced platelet retention in glass bead columns. They did not induce platelet aggregation but increased the sensitivity to ADP and collagen markedly soon after blood sampling. The addition of small amounts of tissue extracts to PRP, which aggregated spontaneously in PAT III led to spontaneous aggregation 10 minutes after blood sampling. No such effect was observed with spontaneously non-aggregating PRP. Apparently the tissue extracts shortened the time dependent changes in aggregation response from 30 – 90 to less than 10 minutes. The lipoprotein responsible for these effects probably stimulates primary hemostasis and is therefore named “Hemostasis Activating Factor” (HAF).

Neurohumoral effects of ramipril in angina pectoris: A double-blind, placebo-controlled trial against isosorbide dinitrate and the combination of ramipril plus isosorbide dinitrate

Abstract Elevated levels of neurohormones are directly correlated with cardiovascular risk. This double-blind, parallel-group study was designed to examine whether treatment with an angiotensin-converting enzyme (ACE) inhibitor, ramipril, could positively influence the plasma levels of neurohormones—plasma renin activity (PRA), aldosterone, atrial natriuretic peptide (ANP), and endothelin—and whether differences exist between treatments with ramipril, isosorbide dinitrate (ISDN) alone, ramipril + ISDN or placebo. Thirty-two patients (1 woman, 31 men) with congestive heart failure, aged 42 to 72 years (mean, 62.8 years), were enrolled in four parallel study groups. Test medications were administered as a single dose in a double-blind, parallel-group manner with a double-dummy technique as follows: ramipril (5 mg), ISDN (20 mg), the combination ramipril + ISDN, and placebo. PRA, aldosterone, ANP, and endothelin concentrations were estimated before and 1, 2, 3, 4, 6, 8, and 24 hours after drug administration. Plasma endothelin levels decreased significantly in the groups receiving ramipril treatment, whereas no change was seen after placebo or ISDN administration. No significant changes were seen for ANP, whereas PRA increased and aldosterone decreased in the groups receiving ramipril. A single dose of ramipril reduced plasma endothelin in patients with angina pectoris, whereas no significant changes were observed for ANP. The changes seen with PRA and aldosterone were as expected for ACE inhibitors.

On the Mechanism of Platelet Activation During Hemostasis and Thrombosis and on the Effects of Platelet Inhibiting Drugs

Platelets are essentially involved in primary hemostasis. During the first seconds after a vascular lesion they are stimulated on the damaged vessel wall as well as inside and outside of the vessel. Many of today’s concepts on the mechanism of primary hemostasis and also on the first steps in thrombus formation have been derived from aggregometer studies. But it has to be kept in mind that induced or spontaneous aggregation, as they are usually studied in vitro, only occur if the platelets are partially or totally shape changed, have become more adhesive and tend to aggregate much more than the circulating disc-like platelets.

Die Stimulation der Thrombozyten durch Homogenisate aus subcutanem Fettgewebe. Ein Auslöser der Hämostasereaktion

Die primare Blutstillung nach einer Gefasverletzung hangt in erster Linie von einer ausreichenden Zahl und Funktion der Thrombozyten ab. Die heutigen Vorstellungen uber die ersten Schritte der primaren Hamostase gehen zum grosen Teil von in vitro-Befunden aus. So nimmt man an, dafi die Plattchen an der verletzten Basalmembran und an freiliegenden Bindegewebs- und Kollagenfasern haften und sich dort umwandeln, In-haltsstoffe freisetzen und aggregieren. In vivo zirkulieren die Plattchen als flache, konkave Scheiben. Nach der Blutentnahme wandeln sie sich in Zitrat-, EDTA- oder Heparinblut urn, indem sie Fortsatze bllden und kugelformig werden. Dieser Vorgang lauft bei 37°C verhaltnismasig langsam ab - nach 30 min sind ca. 40% der Plattchen formverandert (Breddin et al., 1976). Bei der Untersuchung des Formwandels der Plattchen unmittelbar nach der Blutentnahme machten wir folgende Beobachtungen: Im sofort nach Entnahme ftxierten Zitrat- oder Vollblut einzelner Versuchspersonen fanden wir einen hochgradig gesteigerten Antell der morphologisch veranderten Plattchen von etwa 80% gegenuber dem normalen Ausgangswert von 20%. N ach gezielter Fehlpunktion und Aspiration von Gewebssaft ergab sich bei 10 Probanden eine Stimulationsrate von im Mittel 64,5%. Otfensichtlich ist es moglich, durch Beimengung von Gewebssaft einen Formwandel der Thrombozyten innerhalb von Sekunden zu erzielen. Handelt es sich dabei urn thromboplastisches Material?