Myocardial ischaemia induces platelet activation with adverse electrophysiological and arrhythmogenic effects.

OBJECTIVE: The aim was to investigate how platelet activation during myocardial ischaemia can induce electrophysiological and arrhythmogenic effects, and examine the involvement of different platelet membrane receptors in producing these effects. METHODS: Transmembrane action potentials and electrograms were recorded from isolated, Langendorff perfused guinea pig hearts during normal perfusion, global myocardial ischaemia, and reperfusion during infusion of human platelets. Platelet reactivity was altered by treating platelets with forskolin, aspirin, the platelet activating factor (PAF) receptor antagonist BN 52021, the thromboxane A2 (TP) receptor antagonist GR 32191B, and the alpha 2 adrenoceptor antagonist yohimbine. Myocardial catecholamine depletion was induced by treatment with 6-hydroxydopamine. RESULTS: Platelet infusion had no electrophysiological effects during normal perfusion, but during ischaemia it enhanced the reduction in action potential duration at 95% repolarisation [APD95, 110(SEM 3) ms v 121(5) ms, p < 0.05, at 15 min] and increased the incidence of ventricular arrhythmias (from 56% to 94%, p = 0.04) compared to hearts receiving buffer but no platelets. The reductions in APD95 and the arrhythmogenic effects were attenuated when forskolin treated, aspirin treated or GR 32191B treated platelets were infused (VF: 50% v 94%, p = 0.03; 50% v 94%, p = 0.02; 22% v 94%, p < 0.001, respectively). Similar results were obtained when normal platelets were infused into catecholamine depleted hearts (VF: 60% v 94%, p = 0.0549). These differences were associated with inhibited aggregatory responses to thrombin (for forskolin treated platelets) and the thromboxane mimetic U44069 (for GR 32191B treated platelets). Yohimbine was antiarrhythmic in the presence and absence of platelets, suggesting direct myocardial effects, but BN 52021 had no antiarrhythmic effects. CONCLUSIONS: Myocardial ischaemia causes platelet activation resulting in electrophysiological and arrhythmogenic effects. PAF receptor antagonism does not prevent these effects, but inhibition of platelet reactivity, platelet thromboxane receptor antagonism, and myocardial catecholamine depletion are effective. These findings suggest that the arrhythmogenic effects of platelet activation during myocardial ischaemia are principally mediated by a thromboxane dependent mechanism, while catecholamine release has a contributory role.

Antithrombin III "Northwick Park": a variant antithrombin with normal affinity for heparin but reduced heparin cofactor activity.

Further studies have been carried out in a previously reported family with congenital antithrombin III (AT III) deficiency due to an abnormal variant of AT III (AT III Northwick Park). The variant has been identified in five members of the family, three of whom had a history of venous thrombosis. Inheritance followed an autosomal dominant pattern. The affected family members have reduced levels of antithrombin heparin cofactor (41-67%) and progressive antithrombin activity (44-62%) but normal levels of immunoreactive AT III (91-162%). Two dimensional immunoelectrophoresis (2 DIE) of AT III in the absence of heparin revealed an abnormal fast-moving peak in addition to the normal peak but 2 DIE in the presence of heparin appeared normal. Further studies confirmed that the abnormal AT III binds completely to heparin but has no heparin cofactor or progressive antithrombin activity. These results would be consistent with a mutation affecting the binding site for thrombin.

Haemostasis in normal pregnancy.

A study has been undertaken in 72 women to provide systematic information on the changes that occur in a wide range of haemostatic variables during and after pregnancy. Factors VII, VIII:C, VIIIR:Ag, X, fibrinogen and alpha 1-antitrypsin, rose markedly throughout pregnancy. Factors II and V and alpha 2 macroglobulin all rose early on but then decreased steadily. Antithrombin III:C and Ag fell slightly. There was a marked decrease in fibrinolytic activity from 11-15 weeks onwards. Levels of fibrin degradation products rose from 21-25 weeks onwards. The rise in coagulation factors that occurs could be due to increased synthesis or increased activation by thrombin, or to both. The findings are consistent with a mild degree of local intravascular coagulation from early on in pregnancy in some women.

Analysis of the procoagulant activities of factor IX concentrates.

The Factor VIII content of Factor IX concentrates was investigated by agarose gel electrophoresis which removed the interfering effects of stabilisers and proteolytic enzyme inhibitors. Factor VIII coagulant activity (FVIII C) as measured by clotting and amidolytic methods correlated well with the distribution of FVIII coagulant antigen (FVIII CAg). The FVIIIC activity was apparently not entirely due to the presence of activated coagulation enzymes, since none of the enzymicity patterns observed with various chromogenic substrates correlated completely with the FVIII peak. However, some enzyme activity was detected in positions coincident with the margins of the FVIII peak; these activities may represent complexes of some of the FVIII with activated coagulation factors.

The effect of physiological levels of fibrinogen on platelet aggregation.

Results from the Northwick Park Heart Study (NPHS) suggest that physiological levels of plasma fibrinogen may influence platelet aggregability. This possibility has been further studied by the addition of purified fibrinogen to the blood of 17 study participants with low plasma fibrinogen levels. The results, which were highly consistent between different individuals, showed that fibrinogen increases aggregability as measured by the ADP ED50, the dose of adenosine diphosphate at which aggregation proceeds at half its maximum velocity. However, an increasing plasma fibrinogen level was associated with decreasing aggregability measured by another parameter, the ADP EMR (estimated maximum response). Although the balance of evidence is that the plasma fibrinogen level enhances aggregability, these conflicting results emphasize the limitation of any simple concept of "platelet aggregability".

An overview of current quality control procedures in platelet storage lesion and transfusion.

In brief the major tasks in quality monitoring of platelet concentrates are: (i) To ensure that the process is able to meet the product specifications. (ii) To steer the apheresis session and production laboratory towards prevention-oriented decisions. (iii) To provide data on where problems are likely to occur, helping to identify cause-and-effect relationships. (iv) To communicate information in rapid, accurate and simple ways to help both in the concept of pre-release testing and preventative maintenance. These can only be achieved by exchange of ideas and collaborative work between producer and user but, for the purpose of pre-release testing, the dMPV provides unique criteria for control of platelet concentrates by both users and producer and is a method of choice. The three criteria of acceptability--platelet number, platelet function and leucocyte count--can be effectively measured by one test on paired samples with excellent accuracy using a cell counter, helping to standardize quality determination. In several blood transfusions services a trend in this direction is already on the way.

Quality monitoring of haemapheresis platelet concentrates: sampling in EDTA helps with the standardization and improves the consistency.

At the North London Blood Transfusion Centre, all haemapheresis platelet concentrates (PC) are tested in duplicate by a Technicon H-1 analyser to provide accurate information on the cell separator performance and on the cellular content of packs before issue. Repeated counting on fresh samples (within 4 h) reveals spontaneous aggregation (SA), which causes inconsistency in the estimated platelet yields and invalidates the cellular indices. Preliminary work showed that sampling into EDTA eliminated SA. Further studies on the effect of EDTA were undertaken as follows: (i) using the same PCS preparations (n = 7) stored conventionally and sampled daily in tubes with and without EDTA, and (ii) using fresh V50 and CS3000 PC to assess the difference on samples routinely collected in EDTA tubes for 2 months compared to those collected without EDTA in the previous 2 months. EDTA improved concordance of duplicate counts in fresh products. The increase in platelet yield in V50 and CS3000 preparations (12-22% respectively) was associated with a concomitant decrease (60-74%) in erythrocyte contamination. The variation in leucocyte count (3-5%) was less pronounced but the percentage differential was affected. There was also a systematic increase (8-11%) in mean platelet volume (MPV) due to EDTA. In PCS preparations the EDTA-induced variation in the cellular indices remained essentially in the same order (2-6%), while the MPV decreased progressively on storage. The difference in MPV can be used to assess the storage stability of various PC preparations. The implication of a double sampling technique (CPDA-1 and EDTA/CPDA-1) in the quality monitoring of haemapheresis procedures is substantial.

An overview of current trends in platelet preparation, storage and transfusion.

We present an overview of issues relating to preparation, storage and transfusion of platelet concentrates. The concepts of quality are described and potential benefits from leucodepletion, UVB irradiation and the use of additive solutions discussed. Emphasis is placed on laboratory evaluation of the storage lesion and in particular morphological changes of platelets during storage.

Monitoring the release of glycocalicin in platelet concentrates by ELISA.

Platelet membrane glycoprotein Ib (GPIb) is one of many GPs on the platelet membrane which contribute to the functional and morphological properties of platelets. A principal function of GPIb is its attachment to von Willebrand Factor (vWF) on injured blood vessels which leads to the adhesion of platelets to these vessels. The binding site to vWF resides on glycocalicin (GC), which is a major segment of GPIb. Glycoprotein Ib is particularly susceptible to centrifugation and storage of platelets. The assessment of GPIb status on platelets, therefore, comprises one of many traditional methods for monitoring the quality of platelets during storage. We have recently developed a novel ELISA to monitor GC levels in the supernatant of platelet concentrates (PCs) during storage. Using this ELISA we observed a progressive rise of GC in PC supernatants during storage. A recent study of citrated PCs with or without EDTA produced similar results, and showed a substantial increase of GC levels in EDTA-treated PCs. The GC ELISA could therefore be used as a novel method to monitor PCs during storage under various conditions.

The use of mean platelet volume for evaluation of quality of platelet concentrates.

Mean platelet volume (MPV) was determined on whole blood and platelet concentrates (PC) prepared from units of the same blood, as well as on samples of venous blood taken from donors before plateletpheresis and on PC collected by Spectra (COBE Laboratories Ltd) and CS-3000 (Baxter Healthcare Ltd) cell separators. The mean (+/- SD) MPV for PC prepared from blood (7.18 +/- 0.76 fl, n = 12) was significantly lower than that for whole blood (8.32 +/- 0.72 fl, P < 0.02) suggesting significant separation of young, large and dense platelets together with the red cells. In contrast, the mean MPV for PC collected with Spectra and CS-3000 cell separators was 8.48 +/- 0.52 fl (n = 20) and 8.94 +/- 0.60 fl (n = 12), respectively, and was significantly higher (P < 0.01) than that determined in venous blood samples of donors taken before plateletpheresis (7.76 +/- 0.74 fl and 8.12 +/- 0.62 fl respectively). This indicates preferential separation of large platelets, which are by inference, of better quality, into PC.

Automated processing of leucocyte-poor platelet concentrates.

In view of transfusion reactions and alloimmunization associated with leucocyte contamination of platelet concentrates (PC), there is a general move towards the production of leuco-poor PC. This goal is currently pursued by the production of various PC using buffy coat and apheresis techniques. Although there is no overall consensus on the meaning of 'leuco-poor', by assuming that this refers to a level of 5-50 x 10(7) leucocytes per PC, we were able to make comparisons with available systems used in Europe. In addition to platelet and white cell counts, other markers of PC quality were assessed in some cases. These included traditional markers (such as hypotonic stress response, pH, and lactate and beta-thromboglobulin levels) and newer markers (such as glycocalicin and plasma von Willebrand factor levels). Our preliminary results showed appreciable differences in platelet and white cell content of PC prepared by various types of apheresis equipment. Appreciable differences in the quality of stored PC were also observed between routine PC (non-leuco-poor and buffy coat and apheresed PC (leuco-poor).

Platelet-mediated alterations in cardiac cellular electrophysiology.

Recent clinical and experimental evidence indicates that platelet activation contributes to the arrhythmogenic effects of myocardial ischaemia, but little is known about the electrophysiological effects produced by controlled platelet activation under conditions of normal perfusion and how these might relate to effects during ischaemia. To investigate this, we studied changes in cardiac cellular electrophysiology and arrhythmogenesis during infusion of platelets [10(8)/ml] in isolated, perfused guinea-pig hearts during normal perfusion and global myocardial ischaemia. Hearts were studied in four groups: group A (n = 4) receiving frozen/thawed (activated) platelets; group B (n = 4) receiving normal platelets in the presence of 10(-9) M platelet activating factor (PAF); group C (n = 9) receiving buffer only during normal perfusion and myocardial ischaemia; group D (n = 9) receiving platelets during normal perfusion and myocardial ischaemia. Infusion of platelets (group D) had no effects during normal perfusion, but activated platelets (group A) decreased action potential duration (APD) from 165 +/- 1 ms to 138 +/- 5 ms (mean +/- SE) at 15 min of normal perfusion (P less than 0.02) and produced ventricular fibrillation (VF) in 3/4 at 21 +/- 1 min. Infusion of platelets in the presence of PAF (group B) produced similar reductions of APD during normal perfusion and VF in 2/4. During ischaemia, platelets (group D) increased the incidence of VF (100% vs 56% group C, P less than 0.05) and enhanced the ischaemia-induced reductions in APD (107 +/- 3 ms vs 121 +/- 5 ms (group C) P less than 0.05 at 15 min).(ABSTRACT TRUNCATED AT 250 WORDS)

Glycoproteins Ib and IIb/IIIa in the quality assessment of platelet concentrates during storage.

We have previously shown that levels of soluble glycocalicin (GC) in plasma supernatants derived from units of platelet concentrates (PC) increase progressively during storage. We now report further studies which show that the levels of both microparticle-bound and soluble GC in PC during storage are influenced by exposure of PC samples to EDTA and treatment of PC packs with ultraviolet B (UVB) irradiation. EDTA leads to a significant increase in the release of microvesicle-bound and soluble GC, while UVB irradiation leads to a dose- and rate-dependent increase in GC release. Paradoxically, UVB leads to an unexpected decrease in supernatant levels of von Willebrand factor (vWf) during storage which contrasts with its increase in untreated, stored PC. Moreover, an increase in GC release during storage is associated with a corresponding decrease in platelet size as determined by measurement of mean platelet volume (MPV) in citrated PC. The GC release is significantly correlated with standard platelet functional tests and other new generation tests such as dMPV and supernatant levels of vWf. In addition, preliminary results show the presence of microparticle-bound and soluble glycoprotein (Gp) IIb/IIIa in the supernatant plasma of stored PC. Our results suggest that supernatant levels of GpIb, GpIIb/IIIa, and vWf, together with alteration in MPV, provide essential new informative parameters for quality assessment of PC.

Characteristics of platelet concentrates, with particular reference to Autopheresis C Plateletcell: correlation between dMPV and other tests for platelet function.

The quality of platelet concentrates (PC) collected by the Autopheresis C cell separator was assessed in two Regional Transfusion Centres taking part in a multicentre study. This study also enabled the assessment of a new simple, rapid test of platelet function and comparison with more established tests, such as aggregation to adenosine diphosphate, as a tool for the quality testing of PC. The new test, based upon the measurement of mean platelet volume using automated haematological cell analysers, is rapid and uses the same samples as those used to estimate the platelet and leucocyte content of the concentrates. The high correlation between this test and the other tests of platelet function used in the study suggests that it is an ideal tool in the quality testing of platelet concentrates.

Effect of cold exposure on platelet concentrates: changes in platelet indices and aggregation states.

Samples from platelet concentrates (filtered/non-filtrated, at the beginning/the end of shelf life) were exposed to 4 degrees C overnight, subsequent to dilution in platelet storage media (PSM) and/or exposure to EDTA to induce shape changes. Paired sampling protocol (+/- EDTA) was used and changes in cellular indices and induced-aggregation states were measured by Technicon H*1. Cold induced changes in platelets as identified by increase in MPV (0.5-0.8 fL for EDTA; 1.5-2.0 fL for citrated samples) with concomitant reverse changes in PDW ranging from 2-13 per cent was observed. Processing, storage and cold exposure also induced disparity between leucocyte peroxidase/basophil counts. This in conjunction with changes in platelet counts and cellular indices upon exposure to EDTA provide a unique new tool for assessing the aggregation states of platelets during processing and storage. Both filtration and dilution in PSM affect platelet storage stability. Platelets which have already undergone shape changes (i.e. exposure to EDTA) responded to a lesser degree to cold exposure. Our findings indicate that platelets's response to cold exposure can be used as a simple, reliable and accurate test for assessment of platelet morphological and function integrities.