Klaus Breddin

Time and temperature dependent changes of ADP- and collagen-induced and “spontaneous” aggregation

Abstract ADP- and collagen-induced aggregation and spontaneous aggregation by means of PAT III markedly change if citrated blood or PRP are stored at different incubation temperatures or/and if the time between blood sampling and testing varies. With increasing time interval between blood sampling and performance of the test response of PRP to ADP or collagen and the extent of aggregation in PAT III increase. In ADP-induced aggregation % disaggregation decreases. In collagen-induced aggregation the steepness of the curves and the maximal amplitudes increase with time. A significant correlation between aggregation in PAT III performed 90 – 180 min after blood sampling and ADP-induced aggregation (% disaggregation), was found. All parameters of collagen-induced aggregation (1 μg/ml PRP) showed a positive correlation with angle alpha 2 of PAT III if induced aggregation was studied 30 – 60 min after blood sampling. Time and temperature between blood sampling and testing should be strictly controlled in clinical aggregometer studies. For the investigation of patients with hemorrhagic disorders a long interval (>90 min) between blood sampling and testing is recommended. If enhanced aggregability is to be evaluated by means of PAT III investigations should not be performed before 60 – 90 min after blood sampling. If induced aggregation (ADP, collagen) is used for this purpose, aggregation should be studied between 30 and 60 min after blood sampling. Room temperature (20°C – 25°C) appears to be the most suited incubation temperature for clinical investigations.

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 When the canula was accidentally inserted into the perivascular tissue and then withdrawn to collect venous blood almost all platelets were sphered and showed pseudopodes.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).

Influence of the time interval after venepuncture and the storage temperature on the uptake of 14C-serotonin by human blood platelets

Abstract The active uptake of 14 C-5-HT into human platelets at 37°C was studied at various times (10–130 min) and at various storage temperatures (4°, 22°, 37°C) after venepuncture. 5-HT uptake was significantly influenced by both variables. There was no direct correlation between 5-HT uptake and storage temperatures, storage time and changes in the pH of PRP, resp. The apparent K m -value for the 5-HT uptake (0.5μM) remained constant. However, the K i -values obtained for different uptake inhibitors at the different experimental conditions indicate the need for exact standardization.

The antithrombotic and anticoagulant effects of a synthetic tripeptide and recombinant hirudin in various animal models.

The pharmacologic activities of two thrombin inhibitors (D-MePhe-Pro-Arg-H, recombinant-hirudin) were compared in two animal models. The antithrombotic effect was investigated in vivo in rabbits using a modified Wessler stasis thrombosis model. During these experiments, blood was drawn for ex vivo testing to determine the coagulation profile and to determine plasma concentrations using pre-constructed calibration curves. A dose-dependent antithrombotic effect was observed for both agents. On an equigravimetric basis (100 micrograms/kg i.v.), r-hirudin showed a stronger antithrombotic effect than the tripeptide, which correlated well with the ex vivo anticoagulant effect. No adverse reactions were observed during this study. In a rabbit ear bleeding model, a dose and time dependent hemorrhagic effect was observed for both agents. Only slight bleeding effects were observed at 1.0 mg/kg dosages. These studies show that the tripeptide D-MePhe-Pro-Arg-H and r-hirudin are specific thrombin inhibitors with potent antithrombotic effects and a high therapeutic (antithrombotic/hemorrhagic) index. Furthermore, the results of these two animal models and ex vivo analyses can be used to determine the therapeutic index of thrombin 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?