P. Olsson

Inhibition of thrombin on surfaces coated with immobilized heparin and heparin-like polysaccharides: a crucial non-thrombogenic principle.

Abstract Surfaces prepared from different sulphated polysaccharides and a polyethylene (PE) surface chemically modified with sulphate groups were examined in respect of their interaction with thrombin. Different fractions of heparin, heparan sulphate, dermatan sulphate, chondroitin sulphate and dextran sulphate were used. All the modified surfaces adsorbed thrombin, which retained its activity towards both fibrinogen and a synthetic chromogenic substrate. The activity was readily neutralized on subsequent exposure to plasma or purified antithrombin III on the surfaces prepared with active heparin, but not on the other modified surfaces. The sulphated PE-surface, but not the heparinized surface showed both thrombin and factor Xa activity after blood exposure in vivo. It is concluded that surfaces with an insufficient capacity to neutralize coagulation enzymes captured on the surface constitute examples of thrombogenic surfaces. The heparinized surface may act as an immobilized anticoagulant by providing sites for adsorption and conditions for subsequent inhibition of thrombin.

Platelet and plasma coagulation compatibility of heparinized and sulphated surfaces

Abstract The degrees of platelet adhesion and activation of the plasma coagulation system were measured separately on unmodified poly-ethylene, sulphated polyethylene and different modifications of heparinized surfaces stabilized with glutardialdehyde. Thrombus formation on the unmodified surfaces was shown to involve activation of both the platelets and the coagulation system. Despite minimal platelet adhesion on the sulphated surface and the most stable heparinized one, surface-induced plasma coagulation was demonstrated. Two slightly different modifications of the heparinglutar surface proved to inhibit platelet adhesion and surfaceinduced plasma coagulation.

The 5-hydroxytryptamine take-up mechanism in normal platelets and platelets from migraine and asthmatic patients

Abstract The initial velocity for 5-HT take-up by platelets was studied in platelet rich plasma from healthy blood donors and patients with migraine or asthma. The kinetic data were analyzed according to Lineweaver-Burk and Eadie-Hofstee. The latter plot revealed only one mechanism for active transport of 5-HT through the outer membrane of the normal platelet. In most of the patients no distinct curves depicting a plain active take-up mechanism could be detected. The findings indicate that 5-HT clearance may be severely disturbed in migraine and asthmatic patients.

Determination of platelet adhesion to polyethylene and heparinized surfaces with the aid of bioluminescence and 51chromium labelled platelets

Abstract A simple, rapid and highly sensitive method, which utilizes the light emission reaction between ATP and the firefly enzyme system luciferin-luciferase, was used to measure the degree of platelet adhesion to polyethylene surfaces after their in vitro contact with blood. The measurements showed a highly significant correlation between the surface content of ATP and the number of surface adhered platelets, which was determined with the aid of 51 Cr-labelled platelets. There was, however, always an additional non-platelet associated contribution of ATP, which was considered to originate from an initial and rapid adhesion of leucocytes. On the glutardialdehyde-stabilized heparin surface, the total surface content of ATP was below 1% in comparison with untreated polyethylene, which demonstrated that no significant adhesion of any cells had occurred. ATP-determinations proved to have a lower detection limit than those based on 51 Cr-labelled platelets.

Mode of Action of Protamine in Regard to Its Circulatory and Respiratory Side Effects

Thirty dogs were subjected to infusions of protamine chloride 30 mg/min. Preceded by a pronounced drop in platelet count, there was 2.5–7 min after the start of the infusion a fall in aortic blood pre

Effect of glycosaminoglycans and antithrombin III on uptake and inhibition of thrombin by the vascular wall

Thrombin binds to and is inactivated by the endothelium. The inactivation is potentiated by plasma. The present investigation was designed to clarify the role of vessel wall glycosaminoglycans (GAG) and plasma antithrombin III (AT III) in the inactivation and binding of thrombin by endothelium. Thrombin was shown to bind to vascular endothelium and artificial surfaces containing GAG:s. The binding could be inhibited on both types of surfaces by pretreating them with protamine. Thrombin bound to endothelium was rapidly inactivated in the presence of plasma but only slowly if the plasma was replaced by AT III, AT III-depleted plasma or a balanced salt solution. It is concluded that thrombin binds to vessel wall GAG:s and is inactivated by the endothelium. Potentiation of the inhibition of the endothelially bound thrombin by plasma is dependent upon presence of AT III but an additional plasma factor is also required.

Veno-venous extracorporeal membrane oxygenation with a heparin-coated system in adult respiratory distress syndrome

Three patients with adult respiratory distress syndrome were treated with veno-venous extracorporeal membrane oxygenation, ECMO, using a heparin-coated system for 8, 12 and 34 days, respectively. Despite extracorporeal blood flow of 4-5 l/min, the patients were ventilator-dependent in the initial period of ECMO. Two of the three patients showed bleeding diatheses despite only slightly elevated activated partial thromboplastin time (APTT). Blood platelet count followed a variable course and serum fibrinogen was normal. Acute pulmonary hypertensive crises, fatal systemic infection, recurrent pneumothorax and plasma leakage from the oxygenators were other main complications during ECMO. Two of the three patients survived, and follow-up showed that severely damaged lungs, if supported in the acute phase, can recover sufficiently to permit normal living.

Interference of acetyl salicylic acid, heparin and fibrinogen degradation products in haemostasis of reptilase-defibrinogenated dogs

Abstract Dogs were defibrinogenated with Reptilase-R. The bleeding was measured in fourteen dogs from skin flap wounds on the third day of Reptilase defibrinogenation. Administration of acetyl salicylic acid, heparin and fibrinogen degradation products significantly increased the bleeding. It is concluded that Reptilase-defibrinogenated animals may well prove suitable for studying the haemostatic function of platelets.

Uptake and inactivation of thrombin by the fresh, glutardialdehyde or heparin treated human umbilical cord vein endothelium

Abstract With the aim to elucidate the non-thrombogenic properties of the vascular endothelium and the lining of a certain arterial substitute, the uptake and inactivation of thrombin by the native and modified human umbilical cord veins were investigated. The modification procedures included variations with glutardialdehyde treatment, heparin exposure and storage in ethyl alcohol. The native and all the modified veins adsorbed substantial amounts of thrombin, which was immobilized in an active state. Following exposure to antithrombin III solution, the adsorbed thrombin was inhibited only on the native veins and those treated with glutardialdehyde, stored in ethyl alcohol, exposed to heparin and again stored in ethyl alcohol.

The effect of various inhibitors on the 5-ht binding and uptake by human platelets

Abstract The effect of various potential inhibitors on the 14C-5-HT-hydroxytryptamine (5-HT) uptake in human platelets was studied in fresh undiluted platelet rich-plasma (PRP). The kinetic data, based on 5-HT concentrations ranging from 0.17 to 1.7 μmol/1, were analyzed according to Lineweaver-Burk , Scatchard and Sips. The inhibitors were: adenosinediphosphate (ADP), 5-HT, acetylsalicylic acid (ASA), dipyridamole, N-ethylmaleimid (NEM) and ouabain. In untreated PRP the mode of 5-HT transport corresponded to one active site. ASA did not interfere with the uptake mechanism. ADP in concentrations insufficient to cause platelet aggregation induced passive diffusion of 5-HT in addition to the active transport. All the other agents caused a disturbance in the active 5-HT transport without adding passive diffusion. The inhibitors were uncompetetive (ouabain, dipyridamole) or increased at rising 14 C-5-HT concentrations (5-HT, NEM, dipyridamole). It is concluded that the 5-HT uptake by platelets resembles a two-substrate reaction.