V. Van Wyk

Influence of Platelet Membrane Sialic Acid and Platelet Associated IgG on Ageing and Sequestration of Blood Platelets in Baboons

Platelets were isolated from blood of baboons and treated with neuraminidase to remove platelet membrane sialic acid, a process which artificially ages the platelets. The platelets were then labelled with 111In and their mean life span, in vivo distribution and sites of sequestration were measured. The effect of removal of sialic acid on the attachment of immunoglobulin to platelets were investigated and related to the sequestration of the platelets by the spleen, liver, and bone marrow. Removal of sialic acid by neuraminidase did not affect the aggregation of platelets by agonists in vitro, nor their sites of sequestration. The removal of 0.51 (median, range 0.01 to 2.10) nmol sialic acid/10(8) platelets shortened their life span by 75 h (median, range 0 to 132) h (n = 19, p < 0.001), and there was an exponential correlation between the shortening of the mean platelet life span and the amount of sialic acid removed. The increase in platelet-associated IgG was 0.112 (median, range 0.007 to 0.309) fg/platelet (n = 25, p < 0.001) after 0.79 (median, range 0.00 to 6.70) nmol sialic acid/10(8) platelets was removed (p < 0.001). There was an exponential correlation between the shortening of mean platelet life span after the removal of sialic acid and the increase in platelet-associated IgG. The results suggest that platelet membrane sialic acid influences ageing of circulating platelets, and that the loss of sialic acid may have exposed a senescent cell antigen that binds IgG on the platelet membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

The in vivo effect in humans of pyridoxal-5′-phosphate on platelet function and blood coagulation

Vitamin B6 has an antithrombotic effect. This, based on the results of in vitro studies, has been attributed to an antiplatelet effect. We assessed the in vivo effect of vitamin B6 by measuring the effect of long-term administration of vitamin B6 on platelet function and blood coagulation. Vitamin B6 (pyridoxine hydrochloride), 100mg twice daily p.o. for fifteen days, was administered to 10 healthy volunteers. The bleeding time was measured before the first dose and 15 days after. A baseline value, the acute effect, chronic effect, and the acute-on-chronic effect of vitamin B6 was estimated by measuring platelet function. The following tests were performed: platelet aggregation induced by collagen, ADP and epinephrine; thromboxane A2 (TxA2)-production and prostacyclin inhibition of ADP-induced aggregation. The effects on the coagulation system were monitored by measuring: the prothrombin time, activated partial thromboplastin time and levels of coagulation factor. Vitamin B6 significantly prolonged the bleeding time from 4.1 +/- 1.1 minutes to 6.8 +/- 1.0 minutes (p = 0.0063). Aggregation of platelets with collagen was slightly but not significantly inhibited. Platelet aggregation induced with the agonists ADP or epinephrine was significantly inhibited by vitamin B6, and the platelets tended to aggregate at a slightly decreased rate. The mean TxA2-production was slightly, but not significantly, decreased. Vitamin B6 had no effect on the sensitivity of platelets to prostacyclin, or on the coagulation system. Our results indicate that the antithrombotic effects of vitamin B6 is limited to inhibition of platelet function; there was no measurable influence on coagulation. The results of this in vivo study are however such that clinical trials are warranted to further assess the efficacy of vitamin B6 as an antiplatelet drug.

Kinetics of indium- 111-labelled platelets in HIV-infected patients with and without associated thrombocytopaenia

Abstract:  Seven to 12% of HIV‐infected patients have thrombocytopaenia. The pathophysiology of the thrombocytopaenia is not clear. It has been variously suggested that it may be caused by an increased peripheral platelet destruction, a defect in platelet production, or by a combination of these. The aim of the study was to elucidate the pathogenesis of HIV‐associated thrombocytopaenia. We determined the mean platelet life span (MPLS) and calculated the turnover of autologous indium‐111‐labelled platelets in 17 HIV‐positive patients, seven with thrombocytopaenia. The sites of sequestration of labelled platelets were quantified. The thrombocytopaenic patients had a very short MPLS (3.0±3.8 h) and a marked increase in platelet production (18.2±12.6× 109/l/h). The majority of these patients (5 of 7) had excessive sequestration of platelets in the spleen. Five of the patients with a normal blood platelet count had a shortened MPLS (109±23 h) and increased platelet turnover (3.8±1.2×109/l/h), i.e. the increased peripheral platelet destruction was compensated for by increased platelet production. The other five patients with a normal platelet count had normal MPLS (195±11 h) and slightly increased platelet production (2.5±0.6× 109/l/h). We conclude that patients with HIV‐associated thrombocytopaenia have increased peripheral platelet destruction. Platelet production is elevated but is insufficient to maintain a normal peripheral platelet count. In these patients platelets are predominantly sequestrated in the spleen. Patients with HIV infection and a normal blood platelet count may also have increased platelet production. This may be an early subclinical phase in the development of full‐blown HIV‐associated thrombocytopaenia.

Low molecular weight heparin as an anticoagulant for in vitro platelet funtion studies

Abstract We evaluated the suitability of low molecular weight (LMW) heparin as an anticoagulant for in vitro platelet function tests in 11 normal volunteers. Results with citrated platelets were considered as the standard. Spontaneous platelet aggregation and the aggregation responses to ADP, epinephrine, collagen, ristocetin and thrombin were measured turbid imetrically in an aggregometer. Dose-response and dose-rate curves were constructed for ADP- and epinephrine-induced aggregation. The maximum aggregation response (ED max ) and rate (EDR max ), and the estimated dose of agonist to induce 50% of the maximum response (ED 50 ) and rate (EDR 50 ) were calculated from these curves. The inhibition of ADP-induced aggregation with PGI 2 was expressed as per cent inhibition. The release of ATP and TxA 2 from platelets aggregated with collagen was measured. No spontaneous aggregation occured with either anticoagulant. The ED 50 and the EDR 50 for heparinized platelets were significantly lower for ADP induced aggregation (0.8±0.3μM vs 2.1±1.0μM [p=0.001] and 0.4±0.1μM vs 0.8±0.3μM [p=0.003]). The EDR max with ADP was significantly higher (p=0.004) for heparinized platelets (64±17.0 units/ml vs 50.4±7.6 units/ml). The heparinized platelets aggregated slighty, but significantly, less in response to ristocetin than the citrated platelets. The response of washed heparinized and citrated platelets to thrombin was not significantly different. The per cent inhibition of ADP aggregation with PGI 2 , was significantly lower with heparinized platelets. The release of TxA 2 and ATP was similar for both anticoagulants. These results indicate that LMW heparin is a satisfactory anticoagulant for platelet aggregation tests.

A formula for correcting for the in vitro release of platelet beta- thromboglobulin

The interpretation of platelet beta-thromboglobulin (BTG) and platelet factor 4 (PF4) levels as indicators of in vivo platelet activation is complicated by the artefactual release of these proteins in vitro. A formula was devised to correct for in vitro platelet activation and release of BTG. Blood was collected from normal volunteers by an ideal method and BTG and PF4 levels determined by radioimmunoassay; these were the reference values. Blood from normal volunteers was activated in vitro by standing at room temperature. The BTG and PF4 released was measured at different time intervals. The relationship between BTG and PF4 released was measured at different time intervals. The relationship between BTG and PF4 was measured mathematically best described by a second degree polynomial function. The true plasma BTG value was then calculated by correcting for in vitro release by the general formula: BTG corrected = BTG measured - BtG for PF4 measured + BtG for PF4 reference The validity of the correction formula was tested in 10 normal subjects and in patients with either recent myocardial infarction(n = 10), familial hypercholesterolaemia(n = 10) or arterial prostheses(n = 14). Correction was adequate in normal subjects if the plasma BTG levels did not exceed 260ng/ml. In patients with a thrombotic tendency, the formula overcorrected for in vitro release. This could be ascribed to increased in vivo PF4 levels in these patients, especially those with prostheses. The reference values for PF4 in these patients, and especially those with vascular prostheses, were also higher than normal. The PF4 measured in their plasma thus reflects both in vivo and in vitro released protein. The hypothesis on which the correction formula was based, is therefore not always applicable. It may be possible to improve the correction by establishing formulae for specific disease groups.

Determination of the Dosage of Recombinant Hirudin to Inhibit Arterial Thrombosis in Baboons

Recombinant hirudin, a potent and direct inhibitor of thrombin, effectively inhibits platelet-dependent thrombosis. Our aim was to establish the plasma concentration at which r-hirudin expresses its optimal antithrombotic effect. We measured the extent of inhibition of (111)In-labeled platelet deposition onto 0.6 cm(2) segments of Dacron vascular grafts. These grafts were incorporated as extension segments into exteriorized permanent femoral arteriovenous shunts in baboons. In six control studies a mean of 1.99 +/- 0.26 x 10(9) platelets were deposited at the end of 120 min. In the treatment studies, a thrombus was allowed to form for 10 min in six animals. Treatment for 30 min with r-hirudin at dosages of 140, 70, and 35 microgram/kg/min, but not 14 microgram/kg/min, dose dependently interrupted platelet deposition. The relationship between the percent inhibition of platelet deposition caused by r-hirudin and the plasma concentration of hirudin was exponential (i.e., % Inhibition = 95(1-e(0.23 x [r-hirudin])) (R(2) = 0.76). From this, we estimated that 50% inhibition of platelet deposition will occur at a plasma concentration of approximately 3.3 microgram r-hirudin/mL and 80% at 8.1 microgram/mL. The relationship between the inhibition of platelet deposition and the plasma concentration of hirudin makes it possible to estimate the dose of hirudin that will result in a given level of inhibition of platelet deposition.

Dose-Dependent Inhibition of Acute Arterial Thrombosis by Monoclonal Antibody (16N7C2) in a Baboon Model

The pivotal role that blood platelets play in haemostasis and thrombosis caused a keen interest in the development of specific inhibitors of platelet function. The platelet glycoprotein (GP)IIb/IIIa receptor complex for fibrinogen is a particularly attractive target for therapeutic intervention, since it is the exclusive mediator of platelet aggregation. There are about 50,000 GPIIb/IIIa receptors on the surface of normal platelets. When they are reduced to about 10,000 there is a marked decrease in platelet aggregation, a mildly prolonged bleeding time and abolition of in vivo thrombus formation1. 16N7C2, a murine monoclonal antibody against human platelets, was developed at the Center for Thrombosis and Vascular Research at the University of Leuven. Preliminary studies showed that it blocks the GPIIb/IIIa receptors of human and baboon platelets, but that it has no effect on cat, dog, pig, rabbit, rat, hamster, mouse and guinea-pig platelets. We therefore decided to evaluate the in vivo potential of this monoclonal antibody in baboons.