Iron-induced platelet aggregation measurement: a novel method to measure platelet function in stenting for ST segment elevation myocardial infarction

Background Iron and (stainless) steel are potent platelet aggregation activators, and may be involved in stent thrombosis, a serious complication after intracoronary stenting. Current platelet function tests are suboptimal, because of inappropriate agonists and/or lack of reproducibility. We tested the feasibility and reproducibility of a novel platelet function test using stainless steel as an agonist and compared it with other platelet function tests. Materials and methods In 111 patients with acute ST segment elevation myocardial infarction (STEMI), duplo measurements of iron (Fe)‐induced platelet aggregation (FIPA) were performed after clopidogrel, acetylsalicylic acid and/or tirofiban treatment. Within 1 h, citrated blood samples drawn from the femoral sheath before primary percutaneous coronary intervention were added to 100 mg of low carbon steel and after 5 s mixing with vortex, the samples were incubated for 15 min. The ratio between the non‐aggregated platelets in the agonist sample and platelets in a reference sample was calculated as the platelet aggregation inhibition. Results FIPA measurement was highly reproducible (correlation coefficient (R) = 0·942, P < 0·001 between duplo samples). FIPA correlated well with adenosine diphosphate‐induced platelet aggregation (R = 0·83, P < 0·001) but weakly with platelet function analyser‐100 bleeding time (R = 0·56, P < 0·001). FIPA could be measured in patients in which platelet aggregation could not be measured by platelet function analyser‐100 or after adenosine diphosphate. Conclusion This study showed good reproducibility of a novel platelet function test using stainless steel as an agonist and showed correlation with validated platelet function tests. We found that the novel platelet function test is a suitable test for measurement of platelet aggregation inhibition in patients undergoing stenting for STEMI, even when they are taking multiple antiplatelet regimens.     

Obituaries

Platelet aggregation by adenosine diphosphate (ADP) is a self-limited and reversible process. An application of this phenomenon is described which allows removal of the platelets from large volumes of fresh platelet rich plasma (PRP) for the preparation of platelet concentrates. The macroscopic platelet clumps resulting from a concentration of 10 μgm ADP per ml of PRP are removed by centrifugation at 50 × g for 10 minutes. Resuspension of these platelets in 20 cc of native plasma results in a platelet concentrate that is 80 to 90 per cent as effective per unit as PRP in its ability to elevate the platelet count in recipients. Such concentrates are superior to concentrates prepared by other methods. The posttransfusion survival of ADP platelets compares favorably with the survival of platelets administered as PRP. There is evidence of minor sequestration but there is no apparent irreversible damage to platelets handled in this manner. Alkaline plasma and increase in plasma ionized calcium enhance the ADP aggregation and improve the efficiency of in vitro separation of platelets from PRP. However, the resulting concentrate is less effective in vivo, because of prolonged and slowly reversible clumping, and failure of these platelets to circulate.     

Impaired platelet prostacyclin receptor activity: a monozygotic twin study discordant for spinal cord injury

Coronary artery disease (CAD) has been reported to occur prematurely in individuals with spinal cord injury (SCI). Although persons with SCI have metabolic abnormalities that may predispose them to CAD, other potential aetiologies may also be operative. Increased platelet aggregation, among other factors, initiates thrombus formation at the site of the vessel injury, which may acutely obstruct arterial blood flow. Prostacyclin is known to have a beneficial effect to inhibit platelet aggregation and prevent thrombus formation. Platelets were studied from 12 pairs of monozygotic twins, one co‐twin with SCI. Each twin pair had similar patterns of platelet aggregation with adenosine diphosphate (ADP), thrombin or collagen, as well as inhibition of platelet aggregation by prostacyclin (PGE₁/I₂) and synthesis of cyclic adenosine mono phosphate (AMP) by the prostanoid. However, the twin pairs differed in their response to PGE₁/I₂ inhibition of platelet‐stimulated thrombin generation that was completely inhibited in non‐SCI platelets but not in SCI platelets. Scatchard analysis of the binding of ³H‐prostaglandin E₁, a stable prostacyclin receptor probe, showed the presence of one high‐affinity (K_d1=8·1 ± 2·8 nM ; n_l=168 ± 35 sites per platelet) and one low‐affinity (K_d2=1·1 ± 0·22 μM ; n₂=1772 ± 220 sites per cell) prostacyclin receptor in normal platelets, whereas in SCI platelets there was a significant loss (P<0·00l) of high‐affinity receptor sites (K_d1=6·34 ± 1·80 nM ; n₁=42 ± 11 sites per platelet) with no significant change in the low‐affinity receptor sites (K_d2=1·2 ± 0·23 μM ; n₂=1860 ± 412 sites per cell). These discordant platelet findings in identical twin pairs raises the possibility of an environmental aetiology for accelerated CAD in those with SCI. The loss of inhibitory effect of PGI₂ on thrombin generation in the twin with SCI appears to be because of loss of platelet high‐affinity prostanoid receptors, which may contribute to atherogenesis in individuals with SCI.     

Standardization of light transmittance aggregometry for monitoring antiplatelet therapy: an adjustment for platelet count is not necessary

Summary. Background: Light transmittance aggregometry (LTA) is considered to be the ‘gold standard’ of platelet function testing. As LTA has been poorly standardized, we analyzed the results of LTA in healthy subjects and patients with antiplatelet therapy using different concentrations of agonists and performing tests in non‐adjusted and platelet count‐adjusted platelet‐rich plasma (PRP). Methods: LTA was performed in 20 healthy subjects and in patients treated with aspirin (n = 30) or clopidogrel (n = 30) monotherapy, as well as in patients on combination therapy (n = 20), using arachidonic acid (ARA 0.25 and 0.5 mg mL⁻¹) and adenosine diphosphate (ADP 2 and 5 μm ) as agonists and performing platelet function tests in non‐adjusted and platelet count (250 nL⁻¹ ± 10%)‐adjusted PRP. Results: The overall platelet aggregation response is decreased after adjusting the PRP for platelet count compared with measurements in unadjusted PRP. The variability of aggregation results is high in adjusted PRP in the subgroup of healthy subjects, ranging from 9.2–95.3% (5th–95th percentile) relative to 77.6–95.5% in non‐adjusted PRP when determining maximum aggregation to ARA 0.5 mg mL⁻¹. Late aggregation using ADP 2 μm ranges from 3.8–89.9% in adjusted PRP compared with 42.9–92.5% in non‐adjusted PRP. Maximum aggregation using ARA 0.5 mg mL⁻¹ in non‐adjusted PRP differentiates between aspirin‐treated patients and healthy controls well, whereas late aggregation using ADP 2 μm in non‐adjusted PRP offers the best discrimination between clopidogrel‐treated patients and healthy controls. Conclusion: Adjustment of PRP for platelet count does not provide any advantage and therefore the time‐consuming process of platelet count adjustment is not necessary.     

Whole-body cooling increases plasma endothelin-1 levels in women with primary Raynaud's phenomenon

To understand better the role of endothelin-1 (ET-1) in the pathogenesis of primary Raynaud's phenomenon (PRP), we investigated the basal ET-1 plasma levels and changes after whole-body cooling in healthy women and those with PRP. The study was performed as an open parallel-group comparison during the month of February. The Raynaud group included 21 female patients (mean age 45·3 years, range 21–57 years) who had had disabling Raynaud's phenomenon for a mean period of 17 years (range 2–26 years). The control group consisted of 25 healthy women (mean age 43·6 years, range 27–56 years). Plasma levels of ET-1 were measured on two separate occasions: once after 30 min of rest at room temperature and after 40 min of whole-body cooling. There were no significant differences in baseline plasma ET-1 levels between the two groups of women. The plasma ET-1 levels increased significantly in the PRP group after cold exposure (mean difference 0·11 pmol l^?1, 95% CI 0·005–0·214, P = 0·012). In contrast, the levels of plasma ET-1 in the control group did not change significantly after cold provocation. In conclusion, no differences in plasma basal levels of ET-1 were observed between the two groups. However, women suffering from Raynaud's phenomenon responded with a slight but significant elevation in plasma levels of ET-1 after whole-body cooling, whereas the healthy control subjects did not. The results from the present study confirm previous observations that endothelial dysfunction may be of aetiological importance in PRP.     

Decreased prostacyclin sensitivity of platelets in patients with Behçet''s syndrome

Abstract Patients with Behçet's syndrome have an increased risk of arterial and venous thrombosis, and abnormal platelet function has been implicated. Platelet function was studied in nine patients with Behçet's syndrome and in nine age- and sex-matched healthy volunteers. Platelet aggregation in response to ADP was measured, and the threshold concentration required to produce irreversible aggregation determined. Sensitivity of platelets to the inhibitory effect of prostacyclin was also determined. In addition, plasma levels of the platelet-specific proteins, β-thromboglobulin and platelet factor 4, and stimulated platelet thromboxane B₂ production, were measured. Platelets from patients with Behçet's syndrome showed normal aggregation in response to ADP, irrespective of disease activity. Platelet sensitivity to prostacyclin was, however, decreased compared with controls—with a mean prostacyclin ID₅₀ of 5·5 ± 1·3 ng ml^-1(mean ± SEM) and 1·9 ± 0·3 ng ml^-1, respectively (P < 0·01). This reduction in platelet sensitivity to prostacyclin was greatest in patients with the most active disease. These results suggest that Behçet's syndrome may be associated with altered platelet function, and this may have important consequences with regard to the increased risk of thrombosis associated with this condition.     

The highly specific platelet glycoprotein (GP) VI agonist trowaglerix impaired collagen-induced platelet aggregation ex vivo through matrix metalloproteinase-dependent GPVI shedding

Summary.  Background:  C-type lectin proteins (CLPs) have diverse targets including platelet GPIb, GPVI and integrin α₂β₁, and affect platelet function in a various way. In this study, we characterized a huge, heterodimeric venom protein, trowaglerix, which belongs to the CLP family. Methods:  We purified a potent platelet-aggregation inducer, trowaglerix, from the crude venom of Tropidolaemus wagleri . Biotinylated trowaglerix was used for binding assays, and immunoblotting was used to investigate the signal transduction involved. Results:  Two distinct subunits of trowaglerix with similar masses of around 16 kDa were eluted by high-performance liquid chromatography after reduction and alkylation. Trowaglerix induced platelet aggregation of washed human platelets and platelet-rich plasma (PRP) in a concentration-dependent manner. Biotinylated trowaglerix specifically bound to platelet membrane GPVI, but not to GPIb or α₂ integrin. Treatment with trowaglerix induced GPVI loss in human platelets in vitro and impaired the platelet aggregation of mouse PRP ex vivo in response to collagen but not in response to adenosine diphosphate (ADP). However, GM6001, a matrix metalloproteinase (MMP) inhibitor, inhibited trowaglerix-induced GPVI cleavage and restored the platelet responsiveness of PRP to collagen. Conclusions:  Trowaglerix activates platelets through specific binding to GPVI, leading to kinases-dependent exposure of functional α_IIbβ₃ and platelet aggregation, and also induces MMP-dependent GPVI shedding from platelets.     

Platelet function normalization after a prasugrel loading‐dose: time‐dependent effect of platelet supplementation

Summary. Background: Hemostatic benefits of platelet transfusions in thienopyridine‐treated acute coronary syndrome (ACS) patients may be compromised by residual metabolite in circulation.Objectives: To estimate the earliest time after a prasugrel loading‐dose when added platelets are no longer inhibited by prasugrel's active metabolite.Methods: Baseline platelet reactivity of healthy subjects (n = 25, 30 ± 5 years, 68% male) on ASA 325 mg was tested using maximum platelet aggregation (MPA, ADP 20 μm ) and VerifyNow^® P2Y12 and was followed by a 60 mg prasugrel loading‐dose. At 2, 6, 12 and 24 h post‐dose, fresh concentrated platelets from untreated donors were added ex‐vivo to subjects’ blood, raising platelet counts by 0% (control), 40%, 60% and 80%. To estimate the earliest time when prasugrel's active metabolite's inhibitory effect on the added platelets ceases, platelet function in supplemented samples was compared across time‐points to identify the time when effect of supplementation on platelet function stabilized (i.e. the increase in platelet reactivity was statistically similar to that at the next time‐point).Results: Supplemented samples showed concentration‐dependent increases in platelet reactivity vs. respective controls by both MPA and VerifyNow^® at all assessment time‐points. For each supplementation level, platelet reactivity showed a sharp increase from 2 to 6 h but was stable (P = NS) between 6 and 12 h.Conclusions: The earliest measured time when supplemented platelets were not inhibited by circulating active metabolite of prasugrel was 6 h after a prasugrel loading‐dose. These findings may have important implications for prasugrel‐treated ACS patients requiring platelet transfusions during surgery.     

Large platelets continue to circulate in an activated state after myocardial infarction

Abstract This study was intended to investigate the actual platelet activation status after an acute coronary event. The activation status of circulating platelets was assayed directly by measuring the membrane activation markers CD62 and CD63 with the Düssel-dorf III flow cytometry test in 22 patients with the diagnosis of acute myocardial infarction during the 48-h observation period following the acute event. The number of activated, marker-positive sample platelets was significantly increased in the post-MI patients: CD62: 5·8%× 2·25^±1 vs. 3·5%× 2·32^±1, P≤ 0·05; CD63: 18·7%× 1·77^±1 vs. 4·6%× 2·16^±1, P≤ 0·00·1. The platelet volume and count were concomitantly increased (12·1 ± 2·4 fl/ 236 ± 90 times 10³μl^-1 compared to 8·3 ± 1·6 fl/187 ± 42 times 10³μl^-1) in the control group. Particularly large platelets were identified as being activated documented by the exponential increase in the difference in CD63-binding sites per sample platelet above the 90%-percentile and below the 10%-percentile of the volume distribution: Δ+ 1341 ± 903 (MI patients) vs. Δ+ 276 ± 126 (controls), P≤ 0·00·1. Significant creatine kinase elevation and decrease in platelet count was found in the non-survivor subset (n= 5). We conclude that predominantly large platelets continue to circulate in an activated state after MI. This study provides direct evidence that the assumption of an increased thrombotic potential becomes operative in vivo in MI patients. Besides CK elevation and decrease in platelet count this might possibly constitute a prognostic factor for the short-term outcome of the patients.     

Clot retraction: evaluation in dilute suspensions of platelet-rich plasma and gel-separated platelets.

Suspensions of platelet-rich plasma (PRP) or gel-separated platelets (GSP) can be used to evaluate clot retraction subsequent to platelet aggregation and fibrin formation. PRP (200,000 per cubic millimeter) or GSP (200,000 or 100,000 per cubic millimeter) are diluted 1:10 (PRP) or 1:8 (GSP) in phosphate buffer, pH 7.4, and clotted with a high concentration (2.5 U. per milliliter) of thrombin. Human fibrinogen (25 mg. per cent) is added to GSP prior to dilution. Clot retraction is 91 to 100 per cent completed in 1 hour and is quantified by measurement of residual fluid volume. Test conditions are unfavorable for fibrinolysis. Very low concentrations of fibrin/fibrinogen degradation products D and E are detected in residual fluid, and no erythrocyte fall-out occurs. Furthermore, the extent of retraction in the dilute systems is related only to platelet numbers and platelet function. The dilute PRP and GSP methods allow evaluation of clot retraction in the presence of PGE1, the most potent inhibitor of platelet aggregation induced by conventional concentrations of collagen, ADP, epinephrine, and thrombin (0.1 to 0.5 U. per milliliter). High concentrations of PGE1 (to 6 x 10(-6) M) do not inhibit aggregation of GSP, fibrin formation, or platelet-fibrin interaction induced by 2.5 U. per milliliter of thrombin. In contrast, PGE1 concentrations as low as 1.5 to 3.0 x 10(-8) M inhibit clot retraction in both the dilute PRP and GSP systems. Thus, using dilute PRP or GSP the effects of platelet aggregation inhibitors on clot retraction can be determined independently of effects on platelet aggregation.     

The effect of exercise on platelet beta-adrenoceptor function and platelet aggregation in healthy human volunteers

Summary. Plasma levels of adrenaline and noradrenaline, platelet cyclic-AMP (cAMP) content, platelet aggregation, platelet release of beta-thromboglobulin, and platelet factor 4 and serum content of thromboxane B₂(TXB₂) and 6-keto-PGF_1α were measured in 12 healthy male volunteers (age 38–72, mean 54·2 years) who were tested at rest and immediately after five min light cycle exercise. The plasma levels of adrenaline and noradrenaline increased significantly after exercise (P<0·01). The platelet cAMP level was not changed by exercise. The functional capacity of platelet beta-adrenoceptors, determined as cAMP production after beta-adrenoceptor stimulation in vitro, decreased highly significantly after exercise in all 12 volunteers (P<0·01). No alteration was observed in platelet aggregation induced by adrenaline or in platelet release of beta-thromboglobulin or platelet factor 4. No change was observed in the serum levels of TXB₂ and 6-keto-PGF_1α. In conclusion: light cycle exercise results in a decreased functional capacity of platelet beta-adrenoceptors, but has no effect on platelet aggregation or platelet release. This might indicate a concomitant and equal decreased functional capacity of platelet alpha-adrenoceptors.     

Platelet aggregability in smoking and non-smoking subjects

Summary. The hypothesis was investigated that the arachidonic acid (AA) system has a different impact on platelet function in smoking compared to non-smoking subjects. Arterial blood was sampled from smokers and non-smokers, and platelet-rich plasma (PRP) was prepared. There were no differences in sex and age distribution between the groups. One portion of the PRP was used to determine the lowest amount of AA required to induce platelet aggregation. In other portions the endogenous antiaggregatory (prostacyclin/PGI₂/-like) activity in the blood was determined, after reinforcing it with theophylline. There was no difference between smokers and non-smokers regarding the amount of AA required to induce platelet aggregation. In fresh PRP prepared from blood from non-smoking subjects theophylline (10^-4 M) induced a 12–17% inhibition of the ADP-induced aggregation of platelets, indicating the presence of endogenous sub-threshold concentrations of PGI₂, -like activity in their blood. The corresponding inhibition in fresh PGI₂ prepared from blood from smokers was significantly lower (4–7%), suggesting lower endogenous concentrations of PGI₂-like activity in their blood, or alternatively, decreased platelet sensitivity to the action of such activity. From these data we conclude that smokers differ from non-smokers with regard to their platelet function: platelet aggregability in response to AA is unaffected, while the endogenous anti-aggregatory power in the plasma is decreased. These observations may be of significance for the cardiovascular hazards connected with smoking.     

荜茇酰胺对儿童抗凝、抗血小板聚集作用的体外实验研究

目的观察荜茇提取物荜茇酰胺(PL)对儿童具有抗血小板、抗凝作用,为临床用药提供实验依据。方法抽取30名健康儿童静脉血,随机分为5组,分离富血小板血浆(PRP),取PRP分别加入二甲基亚砜(DMSO)作为空白组,加入阿司匹林(终浓度10μmol/L)作为对照组,加入PL(终浓度分别为20、100、200μmol/L)作为不同浓度PL组,37℃孵育5min,作用后PRP分别加入诱导剂二磷酸腺苷(ADP)终浓度10μmol/L,胶原(Coll)终浓度2.5μg/mL,花生四烯酸(AA)500μg/mL,采用比浊法在37℃条件下测定血小板聚集率。抽取静脉血分离血浆后分5组,将不同浓度PL分别与3组血浆混合,终浓度分别为5、10、20μmol/L,空白组将1%DMSO与血浆混合,对照组将肝素钠与血浆混合(35U/mL),各组37℃孵育5min后上机检测纤维蛋白原(FIB)水平、凝血酶原时间(PT)及凝血酶时间(TT)、活化部分凝血酶原时间(APTT)。结果与对照组比较,PL(20、100、200μmol/L)干预后对AA、Coll诱导的血小板聚集均有显著抑制作用(P0.05),PL 100μmol/L、PL 200μmol/L可以显著抑制ADP诱导的血小板聚集率(P0.05);PL 10μmol/L、PL 20μmol/L干预后可以显著延长儿童血浆的PT、APTT、TT值(P0.05),但FIB未见明显变化。结论 PL能抑制儿童血浆的血小板聚集,抑制抗凝活性。     

Residual platelet thromboxane A2 and prothrombotic effects of erythrocytes are important determinants of aspirin resistance in patients with vascular disease

Background: Permanent inactivation of cyclooxygenase‐1 and inhibition of platelet thromboxane A₂ (TxA₂) constitute the main mechanisms underlying the prevention of vascular disease by aspirin. Methods and Results: We studied platelet TxA₂ synthesis and its impact on platelet reactivity and platelet–erythrocyte [platelet‐rich plasma (PRP)–RBC] interactions in 533 aspirin‐treated patients with vascular disease. Seventy aspirin‐free and 16 aspirin‐treated normal subjects were evaluated as controls. Collagen (1 μg mL^?1)‐induced platelet activation (¹⁴C‐5HT release) and recruitment (proaggregatory activity of cell‐free releasates from activated platelets) were assessed in PRP, PRP + RBC, and whole blood (WB). TxA₂ was quantified in releasates from WB. Aspirin inhibited TxA₂ synthesis and platelet function in all patients, but to different degrees. Forty‐two patients (8%) displayed partial (<95%) inhibition of TxA₂ relative to that of aspirin‐free controls. They produced >3.5 ng mL^?1 TxA₂ and had higher platelet reactivity than 491 patients who had undetectable TxA₂ or produced residual TxA₂ (R‐TxA₂; ≤3.5 ng mL^?1). Patients with R‐TxA₂ were distributed into TxA₂ quartiles. Patients in the third and fourth quartiles had significantly elevated ¹⁴C‐5HT release in PRP, which was markedly amplified in PRP + RBC and WB. TxA₂ in the fourth quartile translated into increased platelet aggregation and recruitment. Significant correlations were found between R‐TxA₂ and platelet hyperfunction. Conclusion: Biochemical markers (TxA₂ synthesis, ¹⁴C‐5HT release) and biological assays (platelet aggregation and recruitment) used to monitor the aspirin effect in a large population of patients presenting with vascular disease have evidenced the importance of R‐TxA₂ and the prothrombotic effects of RBC in aspirin resistance.     

Evaluation of electrical aggregometry: comparison with optical aggregometry, secretion of ATP, and accumulation of radiolabeled platelets

Platelet aggregation has been most commonly studied in vitro by measuring increases in light transmission as platelets aggregate in PRP. Recently, an electrical impedance method for measuring platelet aggregation has been introduced. This method can be used with either PRP or whole blood and measures an increase in impedance across electrodes placed in the blood samples as platelets accumulate on them. We have compared the results obtained by the two methods, using ADP and collagen as aggregating agents, and also have measured the secretion of platelet ATP simultaneously. Although the aggregometry results were similar, recordings obtained by the electrical method did not distinguish two waves of platelet aggregation or correlate with secretion as well as recordings obtained by the optical method. When PGI2 or PGE1 was added to the PRP, both the rate and extent of the increase in light transmittance were inhibited, but the main effect on the increase in impedance was a decrease in its rate and not in its extent. Increases in impedance and secretion of ATP were also measured in whole blood after the platelets had been labeled with a 125I-containing antibody specific for platelet surface glycoproteins. It appeared that the increases in impedance lagged several minutes behind the formation of platelet aggregates and the secretion of platelet ATP. It is suggested that there are advantages and disadvantages to both methods of measurement of platelet aggregation and that the parameters being measured must be clearly understood to properly interpret the results.     

Paired in vivo and in vitro comparison of apheresis and “recovered” platelet concentrates stored for 5 days

Using a paired study, in vivo and in vitro characteristics of apheresis platelets collected on a cell separator and single-donor whole-blood (recovered) platelets via platelet-rich plasma (PRP) were compared after storage for 5 days in similar plastic containers. Autologous platelets from each of 12 volunteers were labeled with ¹¹¹Indium after storage and reinjected. There was no significant difference in circulating recovery between platelets prepared by the two methods, and only one of five models of survival showed a significant difference. Hypotonic shock recovery was significantly better in apheresis than recovered platelets (57.0% and 32.4%, respectively), whilst aggregation to ADP at 3.2 μM and 32 μM was significantly higher in recovered than in apheresis platelets (17.0% and 45.2% versus 7.8% and 32.9%, respectively). Lactate dehydrogenase (LDH) content was significantly higher in recovered platelets (143.3 versus 77.1 IU/10¹¹ platelets), but LDH release was similar (15.0% cf. 12.6%). There was no significant difference between the two platelet preparations for platelet concentration, pH, aggregation with the calcium ionophore A23187 or collagen plus epinephrine, or ATP content or release. $bL-TG release was lower in apheresis platelets. Neither product was consistently better than the other for the parameters tested, but apheresis platelets have the advantage of lower donor exposure to the patient.     

Platelet transfusion refractoriness responding preferentially to single donor aphaeresis platelets compatible for both ABO and HLA

Platelet transfusion refractoriness is challenging to manage. When human leucocyte antigens (HLA)‐sensitized patients fail to respond to HLA‐matched (HLA‐m) platelets, non‐immune destruction may be assumed, and collections of HLA‐m platelets abandoned. We report cases of highly HLA‐sensitized patients whose only satisfactory platelet transfusion responses were consistently associated with products compatible for both HLA‐ and ABO‐matched (HLA‐m/ABO‐m) platelets, and in whom unsatisfactory increments occurred if either form of major incompatibility was permitted (HLA‐u or ABO‐u). Absolute platelet increments (APIs) were measured and classified as satisfactory if ≥10 and unsatisfactory if <10. Patient 1, age 59 years, group O, with myelodysplastic syndrome/acute myelogenous leukemia (MDS/AML), was unresponsive to either fresh ABO‐m or HLA‐m platelets. Of 17 HLA‐m platelets, satisfactory responses occurred for 75% of HLA‐m/ABO‐m units, and failures for 100% of HLA‐m/ABO‐u, with mean API differing significantly (14·1 vs 1·1, P = 0·0059). Of 36 HLA‐m platelets given to patient 2, age 49 years, group O, Gravida 2 Para 2, with severe aplastic anaemia, a satisfactory response occurred with 75% of HLA‐m/ABO‐m units, and failures for 63% of the HLA‐m/ABO‐u (mean API 26·7 vs 7·6, P = 0·008). Increment failures from HLA‐m platelets need not imply intractable refractoriness. If resources permit, selection of HLA‐m/ABO‐m platelets may optimise the incremental response.     

Inihibition of human platelet aggregation by dipyridamole and two related compounds and its modification by acid glycoproteins of human plasma.

The inhibitory effect of dipyridamole (RA 8) and its two derivatives (RA 233 andSH 869) on platelet aggregation in platelet-rich plasma (PRP) AND IN SUSPENSIONSOF WASHED PLATELETS WAS EVALUATED USING 3 AGGRESSING STIMULI: ADP, thrombin, and collagen. Mean effective dose (ED'30) of RA 8 causing 50 percent inhibition of plateletaggregation of washed human platelets by ADP, collagen, or thrombin varied from 1.2 x 10'-7 to 1.8 x 10'-7M. On the other hand, RA 8 caused little inhibition aggregation in human PRP. RA 233 and SH 869 produced similiar degrees of inhibition ofplatelet aggregation in human PRP and in suspensions of washed human platelets.Platelet-poor plasma, fraction VI-acid glycoproteins, or purified alpha'1-acid glycoprotein complex was isolated by means of Sephadex G-25 gel filtration. It is postulated that the formation of this complex leads to the blocking of the capacity of RA 8to inhibit platelet aggragation. RA 233 and SH 869 had little capacity to form complexes with acid glycoproteins of human plasma. This may explain the effectiveness of these compounds in inhibiting platelet aggregation in PRP.     

Bronchopulmonary responses to endothelin-1 in sensitized and challenged guinea-pigs: role of cyclooxygenase metabolites and platelet-activating factor

Summary— The effect of phosphoramidon on the increase in pulmonary inflation pressure (PIP) induced by endothelin-1 (ET-1) administered by aerosol in ovalbumin (OA)-sensitized and challenged guinea-pigs was investigated after pretreatment or not of the animals with the neutral endopeptidase inhibitor, phosphoramidon, the cyclooxygenase inhibitor, indomethacin or the platelet activating factor (PAF) antagonist, BN 50730. When guinea-pigs were pretreated by phosphoramidon (0.1 mM, aerosol for 15 min), a significant enhancement of PIP was observed after administration of ET-1 (1 or 3 μg ml⁻¹, aerosol for 2 min), whereas these doses of the peptide were only slightly active in control animals. In sensitized and unchallenged guinea-pigs, ET-1 (1 or 3 μg·ml⁻¹, aerosol for 2 min) induced, as in controls, a moderate increase in PIP. In contrast, aerosol exposure of OA in sensitized guinea-pigs developed an increased PIP following ET-1 (1 and 3 μg·ml⁻¹, aerosol for 2 min) administration, that was non significantly affected by pretreatment of the animals with phosphoramidon after the dose of 3 μg ml⁻¹ ET-1. Guinea-pigs exposed to phorphoramidon and treated with indomethacin (10 mg kg⁻¹, iv) or BN 50730 (25 mg kg⁻¹, per os) significantly reduced the increase in PIP upon administration of ET-1 (3 μg·ml⁻¹, aerosol for 2 min). No inhibitory effect of indomethacin was noted when ET-1 (3 μg·ml⁻¹, aerosol for 2 min) was administered to sensitized and OA-exposed guinea-pigs, pretreated or not with phosphoramidon. In contrast, BN 50730 significantly reduced the increase in PIP induced by ET-1 observed in sensitized and OA-exposed guinea-pigs. Moreover, this drug was moderately active in reducing the increase in PIP induced by ET-1, when the animals were pretreated by phosphoramidon. These results suggest that a phosphoramidon-sensitive endopeptidase-like enzyme, present in the airway tissue modulates the effect of ET-1. Furthermore, the increase in PIP to ET-1 observed in aerosol-sensitized and antigen-exposed guinea-pigs appears to be mediated by PAF rather than cyclooxygenase metabolites, even though the participation of other mediators in this process is open.