The Jamaican Haemophilia Registry: Describing the burden of disease.

INTRODUCTION: Jamaica has an estimated 200 persons with haemophilia (PWH), who face significant constraints in access to specialized haemophilia care, including access to clotting factor concentrates. AIM: The aim of this paper is to establish the current burden of disease in PWH in Jamaica. METHODS: PWH were enrolled through the University Hospital of the West Indies, Jamaica. The impact of haemophilia was assessed using a comprehensive battery of heath outcome measures that included the following: laboratory, clinical information and validated outcome measures of joint structure and function, activity, and health-related quality of life (HRQoL) to provide a health profile of the Jamaican haemophilia population. RESULTS: In all, 45 PWH were registered (mean age: 29, range: 0.17-69 years), including 13 children (<18 years of age) and 32 adults. In this sample, 41 had haemophilia A (30 severe) and 4 had haemophilia B (3 severe); 10 patients with haemophilia A were inhibitor positive. The results indicate that adults with haemophilia in Jamaica have significant joint damage: mean Haemophilia Joint Health Score (HJHS) = 42.1 (SD = 17.3); moderate activity levels - mean Haemophilia Activities List (HAL) score = 64.8 (SD = 17.8); and low HRQoL scores - mean Haemo-QoL-A score = 62.3 (SD = 19.4). Results for children are also reported but should be interpreted with caution due to the small sample size. CONCLUSIONS: There is a very high burden of disease in PWH in Jamaica. The health profiles reported in this paper are an essential first step in advocating for a multidisciplinary Comprehensive Care Program for assessment and care of PWH in Jamaica.

Normal range of bleeding scores for the ISTH-BAT: adult and pediatric data from the merging project.

Bleeding Assessment Tools (BATs) have been developed to aid in the standardized evaluation of bleeding symptoms. The Vicenza Bleeding Questionnaire (BQ), published in 2005, established a common framework and scoring key that has undergone subsequent modification over the years, culminating in the publication of the ISTH-BAT in 2010. Understanding the normal range of bleeding scores is critical when assessing the utility of a BAT. Within the context of The Merging Project, a bioinformatics system was created to facilitate the merging of legacy data derived from four different (but all Vicenza-based) BATs; the MCMDM1-VWD BQ, the Condensed MCMDM-1VWD BQ, the Pediatric Bleeding Questionnaire and the ISTH-BAT. Data from 1040 normal adults and 328 children were included in the final analysis, which showed that the normal range is 0-3 for adult males, 0-5 for adult females and 0-2 in children for both males and females. Therefore, the cut-off for a positive or abnormal BS is ≥4 in adult males, ≥6 in adult females and ≥3 in children. This information can now be used to objectively assess bleeding symptoms as normal or abnormal in future studies.

In vitro and in vivo stability of diluted recombinant factor VIII for continuous infusion use in haemophilia A.

Factor VIII (FVIII) replacement by continuous infusion (CI) is used postoperatively or after significant bleeding. For young paediatric patients, CI may require FVIII dilution. Variable stabilities of diluted full-length recombinant FVIII Kogenate FS (KG-FS) have been reported under different storage conditions. We investigated the recovery and stability of diluted KG-FS in vitro and in vivo. Kogenate FS was diluted to 50-120 U mL(-1) and its recovery and stability in glass vials or polypropylene syringes was determined. Furthermore, stability of KG-FS diluted to 80 U mL(-1)'administered' via single- and double-pump mock CI systems was tested. Finally, the in vivo stability of KG-FS diluted to approximately 60 U mL(-1) and administered postsurgically by CI with the double-pump to a paediatric patient with severe haemophilia A undergoing implantable venous access device placement was investigated. Initial KG-FS dilution resulted in a 10-20% FVIII loss; a further 25-30% loss occurred over 72 h in vials or syringes. With the double-pump, 1 h recovery was 35%, increasing to 80% by 24 h; the initial losses were because of the Y-infusion of a 10-fold larger volume of saline concomitantly with the FVIII. In vivo, CI resulted in stable FVIII activity levels within the target range. These in vitro results are important for the generation of CI guidelines for diluted KG-FS in the paediatric haemophilic population. That FVIII losses occur upon dilution and with the double-pump does not preclude use of diluted KG-FS. Indeed, stable FVIII levels were maintained when diluted KG-FS was administered by CI with the double-pump to a paediatric patient postsurgically.

Persistence of procoagulant surface expression on activated human platelets: involvement of apoptosis and aminophospholipid translocase activity.

BACKGROUND: Activated platelets express a procoagulant surface when the asymmetric distribution of membrane phospholipids is scrambled, leading to phosphatidylserine (PS) exposure. PS expression, associated with apoptosis in nucleated cells, would be expected to be reversed by aminophospholipid translocase (APLT) activity. OBJECTIVE: To determine whether the procoagulant surface of activated platelets persists after it forms; to examine whether PS expression on platelets is associated with loss of mitochondrial inner membrane potential (DeltaPsi(m)), a hallmark of apoptosis; and to investigate the role of APLT in persistence of PS expression. METHODS: Platelets were stimulated with thrombin, collagen, a combination of both, or the Ca(2+)-ionophore A23187. Up to 4 h after activation, procoagulant surface expression was measured by annexin A5 binding by flow cytometry and by a prothrombinase assay. Flow cytometry was also used to measure PS expression concurrently with DeltaPsi(m) collapse, using CMXRos. APLT activity in annexin A5-negative and -positive platelets was measured flow cytometrically as the percent of 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]caproyl]-sn-glycero-3-phosphatidylserine (NBD-PS) translocated from the outer to the inner membrane leaflet. RESULTS AND CONCLUSIONS: Procoagulant surface expression on activated platelets persisted in vitro for at least 4 h; if such persistence occurs in vivo, there are important implications for the propagation of thrombosis. With the physiological stimuli, only 10-20% of the activated platelets expressed PS on their surface, and of these, only a portion exhibited DeltaPsi(m) collapse, indicating that PS expression can be associated with platelet apoptosis, but can also occur independently. APLT activity was very low in the PS-expressing platelet subpopulation for up to 4 h after activation, indicating that the persistence of a procoagulant surface may be attributed, at least in part, to this reduced APLT activity.

Reduced membrane fluidity in platelets from diabetic patients.

Platelets from diabetic patients are hypersensitive to agonists in vitro. Membrane fluidity modulates cell function, and reduced membrane fluidity in cholesterol-enriched platelets is associated with platelet hypersensitivity to agonists, including thrombin. Decreased membrane fluidity of these platelets is attributed to an increased cholesterol-phospholipid molar ratio in platelet membranes. We examined the response of platelets from diabetic subjects to thrombin, platelet membrane fluidity, and platelet cholesterol-phospholipid molar ratio. Twelve poorly controlled diabetic subjects were compared with 12 age- and sex-matched control subjects. In response to a low concentration of thrombin, mean values for release of [14C]serotonin from washed prelabeled platelets were not significantly different between diabetic and control subjects, but in 8 of 12 diabetic subjects, the release response was greater than in their paired control subjects. Mean steady-state fluorescence polarization values in 1,6-diphenyl-1,3,5-hexatriene-labeled platelets prepared from diabetic subjects were significantly greater than in control subjects; this indicates a decreased membrane fluidity in platelets from diabetic subjects. Total or very-low-density (VLDL), low-density (LDL), or high-density (HDL2, HDL3) lipoprotein cholesterol concentrations in plasma were not significantly different between groups; however, the ratio of VLDL + LDL to HDL2 + HDL3 was significantly greater in diabetic than in control subjects. There was no difference in the total platelet cholesterol-phospholipid molar ratio between groups.(ABSTRACT TRUNCATED AT 250 WORDS)

The activity of the von Willebrand factor cleaving protease ADAMTS-13 in newborn infants.

BACKGROUND: Unusually large von Willebrand factor (VWF) multimers have been observed in patients with thrombotic microangiopathies (TMA), and absence of the VWF cleaving protease ADAMTS-13 activity is considered to be involved in the etiology of TMA. Increased amounts of large multimers of VWF have also been identified in neonates. OBJECTIVE: We assessed ADAMTS-13 activity in healthy neonates, children and adults to establish baseline levels. PATIENTS AND METHODS: Cord blood was collected from 38 full-term newborns; venous samples were taken from 15 neonates on day 2-3 of life. Seventeen children, 24 healthy adults and seven patients with TMA were studied as well. ADAMTS-13 activity was quantified by the binding of the subjects' plasma VWF to collagen before and after enzyme activation. The multimer distribution of VWF was also determined. RESULTS: Neonates and children had percentage ADAMTS-13 activity similar to adults. However, two groups were apparent in the cord blood samples: while 28/38 newborns had percentage activity within the normal range of healthy adults (102 +/- 3.0%), 10 had significantly lower percentage activity (53 +/- 1.1%; P < 0.0001) that normalized by day 2-3. The VWF multimer distribution was the same in all cord blood samples and was not different compared with children and adults. High-molecular-weight VWF multimers were significantly increased in the 2-3-day-old neonates and in TMA patients. CONCLUSIONS: Although ADAMTS-13 activity was similar in neonates compared with adults, 26% of neonates had mildly reduced activity. Further studies are needed to investigate the complex interaction of VWF production and secretion with its size control by ADAMTS-13 in different age groups.

Triple heterozygosity in the integrin alphaIIb subunit in a patient with Glanzmann's thrombasthenia.

We report triple heterozygosity in the integrin alpha(IIb) subunit in a 5-year-old Canadian girl with Glanzmann's thrombasthenia. The patient has a severe bleeding history possibly aggravated by low VWF suggestive of associated type 1 von Willebrand's disease. Platelet aggregation was absent or severely reduced for all physiologic agonists. Flow cytometry showed an approximately 4% residual surface expression of alpha(IIb)beta(3). Western blotting confirmed a low platelet expression of both subunits. PCR-SSCP and direct sequencing showed no abnormalities in the beta(3) gene, but revealed a G-->A transition at a splice site [IVS 19 (+1)] of exon 19 in the alpha(IIb) gene. Of maternal inheritance, the splice site mutation was associated with intermediate levels of alpha(IIb)beta(3) in carriers. Unexpectedly, two G-->A transitions were detected in exon 29 of the alpha(IIb) gene and led to V(951)-->M and A(958)-->T amino acid substitutions. Family studies using restriction enzymes showed that both exon 29 mutations were paternal in origin and cosegregated across three generations. Transient expression in which mutated alpha(IIb) was cotransfected with wild-type beta(3) in COS-7 cells showed that V(951)-->M gave a much reduced surface expression of alpha(IIb)beta(3) and a block in the maturation of pro-alpha(IIb). In contrast, the A(958) substitution appeared to be a novel polymorphism. Our studies highlight an unusual mixture of defects giving rise to severe bleeding in a child and describe the first pathological missense mutation affecting a C-terminal residue of the calf-2 domain of alpha(IIb).

Procoagulant surface exposure and apoptosis in rabbit platelets: association with shortened survival and steady-state senescence.

BACKGROUND: The signal(s) for removal of senescent platelets from the circulation are not fully understood; phosphatidylserine (PS) expression on platelets and another marker of apoptosis, loss of mitochondrial inner membrane potential (DeltaPsim), have been implicated in platelet clearance. OBJECTIVE: To investigate whether shortened platelet survival and steady-state platelet senescence are associated with increased surface exposure of PS and DeltaPsim collapse. METHODS: Survival of in-vitro biotinylated rabbit platelets treated with thrombin or Ca(2+)-ionophore A23187 was tracked by flow cytometry after injection. Steady-state platelet senescence was investigated by infusing biotin to label a platelet cohort. PS expression and DeltaPsim of in-vitro biotinylated platelets and of the aging platelet cohort biotinylated in-vivo were measured by flow cytometry using annexin V-FLUOS and the DeltaPsim-sensitive dye CMXRos, respectively. RESULTS: Although PS expression, DeltaPsim and survival of thrombin-degranulated platelets were similar to those of control platelets, increasing concentrations of A23187 caused increased surface exposure of PS and progressive shortening of platelet survival; only one-sixth of PS-expressing platelets also exhibited DeltaPsim loss. The cohort of senescent, biotinylated platelets remaining in the circulation at 96 h had increased exposure of PS and collapsed DeltaPsim; of the 17% of PS-expressing platelets, one-third did not exhibit DeltaPsim loss. There was also an increase in platelets with collapsed DeltaPsim but not expressing PS. CONCLUSIONS: Platelets with shortened survival and senescent platelets have increased surface exposure of PS, that may be involved in their clearance. PS expression can occur independently of DeltaPsim collapse and conversely, in aged platelets, DeltaPsim loss can occur independently of PS expression.

Effects of dietary sunflowerseed oil and marine oil on platelet membrane fluidity, arterial thrombosis, and platelet responses in rats.

The relationships among platelet membrane fluidity, arterial thrombosis tendency, and platelet aggregation and prostanoid formation were investigated after feeding rats diets enriched in (n-6) or (n-3) polyunsaturated fatty acids. For at least 8 weeks, rats were fed a control diet containing 5 energy % (en%) sunflowerseed oil, a diet high in sunflowerseed oil (50 en%) which contains large amounts of 18:2(n-6), or a diet high in marine oil (5 en% sunflowerseed oil plus 45 en% sperm-whale oil) which contains considerable amounts of 20:5(n-3) and its desaturation and elongation products. Compared with the control diet, platelet membrane fluidity, measured by fluorescence polarization using platelets labelled with 1,6-diphenyl-1,3,5-hexatriene, was significantly increased upon feeding of the sunflowerseed-oil diet or the marine-oil diet. Both of these diets lowered arterial thrombosis tendency, measured by Hornstra's loop technique. Aggregation of platelets in whole blood activated with collagen tended to be higher upon sunflowerseed-oil feeding and lower in the marine-oil group. The concomitant formation of thromboxane A2 was not altered in the sunflowerseed-oil group, but was significantly reduced upon feeding the marine-oil diet. The results indicate that an increase in platelet membrane fluidity may be associated with a reduction in arterial thrombosis tendency. With the techniques used, no direct relationship could be observed among platelet membrane fluidity, platelet aggregation and platelet prostanoid production.

Thrombin binding to platelets from hypercholesterolaemic rats.

Platelets from rats made hypercholesterolaemic with a diet enriched with milk fat and cholesterol and containing taurocholate to promote hypercholesterolaemia aggregated more extensively to a low concentration of thrombin than platelets from rats given a milk fat-enriched diet containing sitosterol. Total and specific binding of thrombin to platelets from hypercholesterolaemic rats was significantly greater than in controls when expressed per mg platelet protein, per mumol platelet cholesterol, or per unit relative surface area. Total and specific binding of thrombin per platelet were not different between the groups. However, platelets from hypercholesterolaemic rats had less protein and cholesterol, were smaller and had less surface area than control platelets; platelet cholesterol content expressed per mg platelet protein was not different. Thus, the increase in thrombin-binding to the smaller platelets from hypercholesterolaemic rats during the first 10 s after its addition may be responsible, at least in part, for the hypersensitivity of these platelets to thrombin.

Platelet hypersensitivity in cholesterol-fed rabbits: enhancement of thromboxane A2-dependent and thrombin-induced, thromboxane A2-independent platelet responses.

Hypercholesterolemia (mean plasma cholesterol: 15 mM) was induced in rabbits by the feeding of a chow diet enriched with a low amount (0.25%, w/w) of cholesterol only. Platelet size and protein content decreased significantly, but the whole blood platelet count did not change. The platelets became enriched in cholesterol, as indicated by a significant increase in the cholesterol:phospholipid molar (C/P) ratio. Specific responses of washed platelets stimulated with various agonists were studied to determine the effects of hypercholesterolemia on the various pathways of platelet aggregation in the absence of plasma components. In platelets from hypercholesterolemic rabbits compared with controls: aggregation induced by ADP was not altered; collagen-induced responses (aggregation, secretion of [14C]serotonin from prelabelled platelets, thromboxane A2 (TXA2) formation, mobilization of [3H]arachidonate from prelabelled platelets) were enhanced; with aspirin-treated platelets, aggregation induced by the TXA2 mimetic U46619 was enhanced: and thrombin-induced responses of both untreated platelets (aggregation, secretion of granule contents, TXA2 formation) and aspirin-treated platelets (aggregation) were enhanced. Thus, platelets from cholesterol-fed rabbits not only form more TXA2, but they aggregate more extensively when stimulated by its mimetic. In addition, it has not been previously recognized that these platelets are also hypersensitive to thrombin-induced aggregation that is independent of TXA2.

Platelet function and survival in rats with genetically determined hypercholesterolaemia.

Platelets from rats with genetically determined hypercholesterolaemia are hypersensitive to aggregation induced by thrombin compared with platelets from their genetic controls without hypercholesterolaemia. Aggregation or release induced by thrombin of platelets from hypercholesterolaemic and control rats correlated significantly with plasma cholesterol concentrations. Platelet responses to ADP or collagen were not different between the groups. The hypersensitivity to thrombin-induced aggregation was independent of released ADP or products of arachidonic acid metabolism. The changes in platelet sensitivity occurred with only moderate increases in plasma cholesterol concentration and with no detectable changes in total platelet cholesterol. The hypersensitivity of platelets from hypercholesterolaemic rats was not associated with a reduction in platelet survival or any significant injury to the aortic endothelium in these animals. Platelets from hypercholesterolaemic rats were smaller than platelets from controls. Thus, platelets from rats with genetically determined hypercholesterolaemia have alterations in function similar to those found with platelets from rats with diet-induced hypercholesterolaemia indicating that this strain can be used to study the mechanisms by which cholesterol can change platelet function without the possible complicating effects of dietary factors. Since platelet hypersensitivity occurred in rats with genetically determined hypercholesterolaemia without a reduction in platelet survival, these studies are also consistent with the reduced platelet survival found in animals with diet-induced hypercholesterolaemia being independent of platelet changes.

Effects of alcohol withdrawal on responses of platelets from alcoholics--a study using platelet-rich plasma from blood anticoagulated with D-phenylalanyl-L-prolyl-L-arginyl chloromethyl ketone (FPRCH2Cl).

Platelet responses stimulated by a range of concentrations of ADP or collagen were studied in platelet-rich plasma (PRP) from alcoholics 24-36 h and 6 days after cessation of drinking, and in PRP from age- and sex-matched controls. The studies were done using plasma from blood anticoagulated with the specific thrombin inhibitor D-phenylalanyl-L-prolyl-L-arginyl chloromethyl ketone (FPRCH2Cl, PPACK); the use of this compound permits the study of platelet responses in plasma at physiological concentrations of ionized calcium. Responses of platelets to ADP (primary aggregation) and collagen (aggregation, secretion of [14C]serotonin from prelabelled platelets, and thromboxane formation) were lower in alcoholics 24-36 h after withdrawal of alcohol compared with controls. This inhibition of platelet function was not due to the presence of alcohol in the blood of the alcoholics. Aggregation in response to ADP did not change during the withdrawal period studied, while collagen-induced aggregation and secretion increased significantly and collagen-induced thromboxane formation tended to increase towards control values. The reduced platelet responses observed in alcoholics and the different rates of "recovery" of different pathways of aggregation towards control values must be due to alterations either in the platelets themselves and/or in the plasma brought about by the chronic presence of ethanol, and its withdrawal.

Effects of ethanol on pathways of platelet aggregation in vitro.

Ethanol, at physiologically tolerable concentrations, did not affect the primary phase of ADP-induced aggregation of human or rabbit platelets, which is not associated with the secretion of granule contents. Potentiation by epinephrine of the primary phase of ADP-induced aggregation of rabbit platelets was also not inhibited by ethanol. However, ethanol did inhibit the secondary phase of ADP-induced aggregation which occurs with human platelets in citrated platelet-rich plasma and is dependent on the formation of thromboxane A2. Inhibition by ethanol of thromboxane production by stimulated platelets is likely due to inhibition of the mobilization of arachidonic acid from membrane phospholipids, as ethanol had little or no effect on aggregation and secretion induced by arachidonic acid or the thromboxane mimetic U46619. Rabbit platelet aggregation and secretion in response to low concentrations of collagen, thrombin, or PAF were inhibited by ethanol. Inhibition of the effects of thrombin and PAF was also observed with aspirin-treated platelets. Thus, in addition to inhibiting the mobilization of arachidonate for thromboxane formation that occurs with most agonists, ethanol can also inhibit aggregation and secretion through other effects on platelet responses.

In vitro effects of ethanol on rabbit platelet aggregation, secretion of granule contents, and cyclic AMP levels in the presence of prostacyclin.

Ethanol, at physiologically tolerable concentrations, inhibits platelet responses to low concentrations of collagen or thrombin, but does not inhibit responses of washed rabbit platelets stimulated with high concentrations of ADP, collagen, or thrombin. However, when platelet responses to high concentrations of collagen or thrombin had been partially inhibited by prostacyclin (PGI2), ethanol had additional inhibitory effects on aggregation and secretion. These effects were also observed with aspirin-treated platelets stimulated with thrombin. Ethanol had no further inhibitory effect on aggregation of platelets stimulated with ADP, or the combination of ADP and epinephrine. Thus, the inhibitory effects of ethanol on platelet responses in the presence of PGI2 were very similar to its inhibitory effects in the absence of PGI2, when platelets were stimulated with lower concentrations of collagen or thrombin. Ethanol did not appear to exert its inhibitory effects by increasing cyclic AMP above basal levels and the additional inhibitory effects of ethanol in the presence of PGI2 did not appear to be brought about by further increases in platelet cyclic AMP levels.

Pretreatment of human platelets with plasmin inhibits responses to thrombin, but potentiates responses to low concentrations of aggregating agents, including the thrombin receptor activating peptide, SFLLRN.

Effects of plasmin on platelets, that influence subsequent responses to aggregating agents, are relevant to attempts to prevent rethrombosis following administration of fibrinolytic agents. We describe plasmin-induced inhibition of platelet responses to thrombin, but potentiation of responses to other aggregating agents. Washed human platelets were labeled with 14C-serotonin, treated for 30 min at 37 degrees C with 0, 0.1 or 0.2 CU/ml of plasmin, followed by aprotinin, washed and resuspended in a Tyrode-albumin solution with apyrase. Incubation with 0.2 CU/ml of plasmin almost completely inhibited thrombin-induced (0.1 U/ml) aggregation, release of 14C-serotonin, and increase in cytosolic [Ca2+]. In contrast, with plasmin-pretreated platelets, aggregation and release of 14C-serotonin were strongly potentiated in response to low concentrations of the thrombin receptor-activating peptide SFLLRN, ADP, platelet-activating factor, collagen, arachidonic acid, the thromboxane mimetic U46619, and the calcium ionophores A23187 and ionomycin. Aspirin or RGDS partially inhibited potentiation. Plasmin-pretreated platelets resuspended in plasma anticoagulated with FPRCH2Cl (PPACK) also showed enhanced responses to aggregating agents other than thrombin. The contrasting effects on responses to thrombin and SFLLRN are noteworthy. Plasmin cleaves GPIIb/IIIa so that it becomes a competent fibrinogen receptor, and binding of 125I-fibrinogen during ADP-induced aggregation was greatly potentiated within 10 s. Potentiation of aggregation by other agonists may be due to increased binding of released fibrinogen. Thus, platelets freed from a thrombus may have increased responsiveness to low concentrations of aggregating agents other than thrombin. These results provide further support for the use of inhibitors of platelet reactions in conjunction with administration of fibrinolytic agents.

Probenecid inhibits platelet responses to aggregating agents in vitro and has a synergistic inhibitory effect with penicillin G.

Probenecid is an anion channel blocker and uricosuric agent, originally developed to slow the rate of excretion of penicillin. It is now also administered with many other drugs to reduce their required dosages. Recently, probenecid (2.5 mM) has been used to prevent leakage of fura-2 or fluo-3 when these indicators of cytosolic Ca2+ levels have been introduced into cells. However, we found that probenecid markedly inhibited the increases in cytosolic Ca2+ caused by ADP, thrombin, the thrombin receptor-activating peptide (SFLLRN, TRAP), ADP, sodium arachidonate, the thromboxane A2 (TXA2) mimetic U46619, and platelet-activating factor (PAF). This finding precluded the use of probenecid with platelets in measurements of cytosolic Ca2+ with indicators such as fura-2. We then investigated the effects of probenecid on aggregation and release of 14C-serotonin from prelabeled platelets. Responses to all the agonists were inhibited by 2.5 mM probenecid, but concentrations as low as 0.25-0.5 mM inhibited responses to agonists that act largely via TXA2 (collagen, sodium arachidonate and U46619). Collagen-induced TXA2 formation was inhibited in a dose-dependent manner. Responses of aspirin-pretreated platelets to thrombin, SFLLRN, U46619 and PAF were also inhibited by probenecid, indicating that prevention of TXA2 formation does not account for all the inhibitory effects. The combination of probenecid with penicillin G produced additive or synergistic inhibition of platelet responses; responses dependent on TXA2 were synergistically inhibited by concentrations of the drugs that are reached in vivo. The synergistic inhibitory effect of probenecid on platelet functions could further impair hemostasis if it has already been partially compromised by the administration of other drugs.

Ethanol inhibits thrombin-induced secretion of the contents of human platelet dense and alpha-granules and lysosomes.

Moderate consumption of alcoholic beverages is associated with a reduction in thromboembolic complications of coronary artery disease, possibly partially attributable to inhibition by ethanol of platelet responses to some aggregating agents. Although ethanol is known to inhibit thrombin-induced secretion of platelet dense granule contents, the effect of ethanol on secretion of alpha-granule and lysosomal contents has not been studied. Using suspensions of washed platelets, and a range of thrombin concentrations (up to 0.1 U/ml), we examined the effect of 87 mM ethanol on secretion of [14C]serotonin from prelabelled platelets as a measure of secretion of dense granule contents. Secretion of alpha-granule and lysosomal contents was examined by flow cytometric measurement of the surface expression of CD62P (P-selectin) and CD63, respectively. Secretion of the lysosomal enzyme, beta-N-acetylglucosaminidase was also quantified. Results were expressed as % of maximum response induced by 1 U/ml thrombin. Ethanol inhibited the thrombin-induced secretion of both dense and alpha-granule contents (P <0.001, 2-way ANOVA), and of lysosomal contents (P <0.005 for CD63 expression and P <0.001 for beta-N-acetylglucosaminidase secretion). When platelets were pretreated with aspirin, thrombin-induced secretion of storage granule and lysosomal contents was slightly inhibited, but secretion was inhibited by ethanol to the same extent as the untreated platelets, indicating that this inhibition was independent of thromboxane A2. Surface expression of CD63 occurred at lower thrombin concentrations than those required for secretion of beta-N-acetylglucosaminidase, possibly due to the presence of some CD63 on granule membranes. Although the role of lysosomal contents in thrombus formation is not established, some constituents of storage granules are known to augment thrombus formation; ethanol's inhibition of their secretion by stimulated platelets may contribute to its beneficial effect on thromboembolism.